Antibodies immunospecific for STEAP1

ABSTRACT

Described is a novel family of cell surface serpentine transmembrane antigens. Two of the proteins in this family are exclusively or predominantly expressed in the prostate, as well as in prostate cancer, and thus members of this family have been termed “STRAP” (Serpentine TRansmembrane Antigens of the Prostate). Four particular human STRAPs are described and characterized herein. The human STRAPs exhibit a high degree of structural conservation among them but show no significant structural homology to any known human proteins. The prototype member of the STRAP family, STRAP-1, appears to be a type IIIa membrane protein expressed predominantly in prostate cells in normal human tissues. Structurally, STRAP-1 is a 339 amino acid protein characterized by a molecular topology of six transmembrane domains and intracellular N- and C-termini, suggesting that it folds in a “serpentine” manner into three extracellular and two intracellular loops. STRAP-1 protein expression is maintained at high levels across various stages of prostate cancer. Moreover, STRAP-1 is highly over-expressed in certain other human cancers.

FIELD OF THE INVENTION

[0001] The invention described herein relates to a family of novel genesand their encoded proteins and tumor antigens, termed STEAPs, and todiagnostic and therapeutic methods and compositions useful in themanagement of various cancers, particularly including prostate cancer,colon cancer, bladder cancer, ovarian cancer and pancreatic cancer.

BACKGROUND OF THE INVENTION

[0002] Cancer is the second leading cause of human death next tocoronary disease. Around the world, millions of people die from cancerevery year. In the United States alone, cancer cause the death of wellover a half-million people each year, with some 1.4 million new casesdiagnosed per year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

[0003] Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the leading causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.

[0004] Generally speaking, the fundamental problem in the management ofthe deadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markers and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

[0005] Recently, there has been a particularly strong interest inidentifying cell surface tumor-specific antigens which might be usefulas targets for various immunotherapeutic or small molecule treatmentstrategies. A large number of such cell-surface antigens have beenreported, and some have proven to be reliably associated with one ormore cancers. Much attention has been focused on the development ofnovel therapeutic strategies which target these antigens. However, fewtruly effective immunological cancer treatments have resulted.

[0006] The use of monoclonal antibodies to tumor-specific orover-expressed antigens in the treatment of solid cancers isinstructive. Although antibody therapy has been well researched for some20 years, only very recently have corresponding pharmaceuticalsmaterialized. One example is the humanized anti-HER2/neu monoclonalantibody, Herceptin, recently approved for use in the treatment ofmetastatic breast cancers overexpressing the HER2/neu receptor. Anotheris the human/mouse chimeric anti-CD20/B cell lymphoma antibody, Rituxan,approved for the treatment of non-Hodgkin's lymphoma. Several otherantibodies are being evaluated for the treatment of cancer in clinicaltrials or in pre-clinical research, including a fully human IgG2monoclonal antibody specific for the epidermal growth factor receptor(Yang et al., 1999, Cancer Res. 59: 1236). Evidently, antibody therapyis finally emerging from a long embryonic phase. Nevertheless, there isstill a very great need for new, more-specific tumor antigens for theapplication of antibody and other biological therapies. In addition,there is a corresponding need for tumor antigens which may be useful asmarkers for antibody-based diagnostic and imaging methods, hopefullyleading to the development of earlier diagnosis and greater prognosticprecision.

[0007] As discussed below, the management of prostate cancer serves as agood example of the limited extent to which molecular biology hastranslated into real progress in the clinic. With limited exceptions,the situation is more or less the same for the other major carcinomasmentioned above.

[0008] Worldwide, prostate cancer is the fourth most prevalent cancer inmen. In North America and Northern Europe, it is by far the most commonmale cancer and is the second leading cause of cancer death in men. Inthe United States alone, well over 40,000 men die annually of thisdisease, second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy remain as the main treatment modalities.Unfortunately, these treatments are clearly ineffective for many.Moreover, these treatments are often associated with significantundesirable consequences.

[0009] On the diagnostic front, the serum PSA assay has been a veryuseful tool. Nevertheless, the specificity and general utility of PSA iswidely regarded as lacking in several respects. Neither PSA testing, norany other test nor biological marker has been proven capable of reliablyidentifying early-stage disease. Similarly, there is no marker availablefor predicting the emergence of the typically fatal metastatic stage ofthe disease. Diagnosis of metastatic prostate cancer is achieved by opensurgical or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy analysis.Clearly, better imaging and other less invasive diagnostic methods offerthe promise of easing the difficulty those procedures place on apatient, as well as improving therapeutic options. However, until thereare prostate tumor markers capable of reliably identifying early-stagedisease, predicting susceptibility to metastasis, and precisely imagingtumors, the management of prostate cancer will continue to be extremelydifficult. Accordingly, more specific molecular tumor markers areclearly needed in the management of prostate cancer.

[0010] There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54:1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252) andprostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95: 1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su et al., 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

SUMMARY OF THE INVENTION

[0011] The present invention relates to a novel family of cell surfaceserpentine transmembrane antigens. Two of the proteins in this familyare exclusively or predominantly expressed in the prostate, as well asin prostate cancer, and thus members of this family have been termed“STEAP” (Six Transmembrane Epithelial Antigen of the Prostate”. Fourparticular human STEAPs are described and characterized herein. Thehuman STEAPs exhibit a high degree of structural conservation among thembut show no significant structural homology to any known human proteins.

[0012] The prototype member of the STEAP family, STEAP-1, appears to bea type IIIa membrane protein expressed predominantly in prostate cellsin normal human tissues. Structurally, STEAP-1 is a 339 amino acidprotein characterized by a molecular topology of six transmembranedomains and intracellular N- and C-termini, suggesting that it folds ina “serpentine” manner into three extracellular and two intracellularloops. STEAP-1 protein expression is maintained at high levels acrossvarious stages of prostate cancer. Moreover, STEAP-1 is highlyover-expressed in certain other human cancers. In particular, cellsurface expression of STEAP-1 has been definitively confirmed in avariety of prostate and prostate cancer cells, bladder cancer cells andcolon cancer cells. These characteristics indicate that STEAP-1 is aspecific cell-surface tumor antigen expressed at high levels inprostate, bladder, colon, and other cancers.

[0013] STEAP-2, STEAP-3 and STEAP-4 are also described herein. All arestructurally related, but show unique expression profiles. STEAP-2, likeSTEAP-1, is prostate-specific in normal human tissues and is alsoexpressed in prostate cancer. In contrast, STEAP-3 and STEAP-4 appear toshow a different restricted expression pattern.

[0014] The invention provides polynucleotides corresponding orcomplementary to all or part of the STEAP genes, mRNAs, and/or codingsequences, preferably in isolated form, including polynucleotidesencoding STEAP proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid,and related molecules, polynucleotides or oligonucleotides complementaryto the STEAP genes or mRNA sequences or parts thereof, andpolynucleotides or oligonucleotides which hybridize to the STEAP genes,mRNAs, or to STEAP-encoding polynucleotides. Also provided are means forisolating cDNAs and the genes encoding STEAPs. Recombinant DNA moleculescontaining STEAP polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of STEAP geneproducts are also provided. The invention further provides STEAPproteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to STEAP proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker, and antibodies conjugated to radionuclides, toxins or othertherapeutic compositions. The invention further provides methods fordetecting the presence of STEAP polynucleotides and proteins in variousbiological samples, as well as methods for identifying cells thatexpress a STEAP. The invention further provides various therapeuticcompositions and strategies for treating prostate cancer, includingparticularly, antibody, vaccine and small molecule therapy.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1. STEAP-1 structure. 1A: Nucleotide and deduced amino acidsequences of STEAP-1 (8P1B4) clone 10 cDNA (SEQ ID NOS. 1 and 2,respectively). The start Methionine is indicated in bold at amino acidresidue position 1 and six putative transmembrane domains are indicatedin bold and are underlined. 1B: Schematic representation of STEAP-1transmembrane orientation; amino acid residues bordering the predictedextracellular domains are indicated and correspond to the numberingscheme of FIG. 1A. 1C: G/C rich 5′ non-coding sequence of the STEAP-1gene (SEQ ID NO. 3) as determined by overlapping sequences of clone 10and clone 3.

[0016]FIG. 2. Predominant expression of STEAP-1 in prostate tissue.First strand cDNA was prepared from 16 normal tissues, the LAPCxenografts (4AD, 4AI and 9AD) and HeLa cells. Normalization wasperformed by PCR using primers to actin and GAPDH. Semi-quantitativePCR, using primers derived from STEAP-1 (8P1D4) cDNA (FIG. 1A), showspredominant expression of STEAP-1 in normal prostate and the LAPCxenografts. The following primers were used to amplify STEAP-1:

[0017] 8P1D4.1 5′ ACTTTGTTGATGACCAGGATTGGA 3′ (SEQ ID NO: 4)

[0018] 8P1D4.2 5′ CAGAACTTCAGCACACACAGGAAC 3′ (SEQ ID NO: 5)

[0019]FIG. 3. Northern blot analyses of STEAP-1 expression in variousnormal human tissues and prostate cancer xenografts, showing predominantexpression of STEAP-1 in prostate tissue.

[0020]FIG. 3A: Two multiple tissue northern blots (Clontech) were probedwith a full length STEAP cDNA clone 10 (FIG. 1A; SEQ ID NO: 1). Sizestandards in kilobases (kb) are indicated on the side. Each lanecontains 2 μg of mRNA that was normalized by using a β-actin probe.

[0021]FIG. 3B: Multiple tissue RNA dot blot (Clontech, Human Master Blotcat#7770-1) probed with STEAP-1 cDNA clone 10 (FIG. 1A; SEQ ID NO: 1),showing approximately five-fold greater expression in prostate relativeto other tissues with significant detectable expression.

[0022]FIG. 4. Nucleotide sequence of STEAP-1 GTH9 clone (SEQ ID NO: 6)corresponding to the 4 kb message on northern blots (FIG. 3A). Thesequence contains an intron of 2399 base pairs relative to the STEAP-1clone 10 sequence of FIG. 1A; coding regions are nucleotides 96-857 and3257-3510 (indicated in bold). The start ATG is in bold and underlined,the STOP codon is in bold and underlined, and the intron-exon boundariesare underlined.

[0023]FIG. 5. Expression of STEAP-1 in prostate and multiple cancer celllines and prostate cancer xenografts. Xenograft and cell line filterswere prepared with 10 μg of total RNA per lane. The blots were analyzedusing the STEAP-1 clone 10 as probe. All RNA samples were normalized byethidium bromide staining and subsequent analysis with a β-actin probe.

[0024]FIG. 5A: Expression in various cancer cell lines and xenograftsand prostate. Lanes as follows: (1) PrEC cells, (2) normal prostatetissue, (3) LAPC-4 AD xenograft, (4) LAPC-4 AI xenograft, (5) LAPC-9 ADxenograft, (6) LAPC-9 AI xenograft, (7) LNCaP cells, (8) PC-3 cells, (9)DU145 cells, (10) PANC-1 cells, (11) BxPC-3 cells, (12) HPAC cells, (13)Capan-1 cells, (14) CACO-2 cells, (15) LOVO cells, (16) T84 cells, (17)COLO-205 cells, (18) KCL-22 cells (acute lymphocytic leukemia, ALL),(19) HT1197 cells, (20) SCABER cells, (21) UM-UC-3 cells, (22) TCCSUPcells, (23) J82 cells, (24) 5637 cells, (25) RD-ES cells (Ewing sarcoma,EWS), (26) CAMA-1 cells, (27) DU4475 cells, (28) MCF-7 cells, (29)MDA-MB-435s cells, (30) NTERA-2 cells, (31) NCCIT cells, (32) TERA-1cells, (33) TERA-2 cells, (34) A431 cells, (35) HeLa cells, (36) OV-1063cells, (37) PA-1 cells, (38) SW 626 cells, (39) CAOV-3 cells.

[0025]FIG. 5B: The expression of STEAP-1 in subcutaneously (sc) grownLAPC xenografts compared to the expression in LAPC-4 and LAPC-9xenografts grown in the tibia (it) of mice.

[0026]FIG. 6. Western blot analysis of STEAP-1 protein expression intissues and multiple cell lines. Western blots of cell lysates preparedfrom prostate cancer xenografts and cell lines were probed with apolyclonal anti-STEAP-1 antibody preparation (see Example 3C fordetails). The samples contain 20 μg of protein and were normalized withanti-Grb-2 probing of the Western blots.

[0027]FIG. 7. Cell surface biotinylation of STEAP-1.

[0028]FIG. 7A: Cell surface biotinylation of 293T cells transfected withvector alone or with vector containing cDNA encoding 6His-taggedSTEAP-1. Cell lysates were immunoprecipitated with specific antibodies,transferred to a membrane and probed with horseradishperoxidase-conjugated streptavidin. Lanes 1-4 and 6 correspond toimmunoprecipitates from lysates prepared from STEAP-1 expressing 293Tcells. Lanes 5 and 7 are immunoprecipitates from vector transfectedcells. The immunoprecipitations were performed using the followingantibodies: (1) sheep non-immune, (2) anti-Large T antigen, (3)anti-CD71 (transferrin receptor), (4) anti-His, (5) anti-His, (6)anti-STEAP-1, (7) anti-STEAP-1.

[0029]FIG. 7B: Prostate cancer (LNCaP, PC-3, DU145), bladder cancer(UM-UC-3, TCCSUP) and colon cancer (LOVO, COLO) cell lines were eitherbiotinylated (+) or not (−) prior to lysis. Western blots ofstreptavidin-gel purified proteins were probed with anti-STEAP-1antibodies. Molecular weight markers are indicated in kilodaltons (kD).

[0030]FIG. 8. Immunohistochemical analysis of STEAP-1 expression usinganti-STEAP-1 polyclonal antibody. Tissues were fixed in 10% formalin andembedded in paraffin. Tissue sections were stained using anti-STEAP-1polyclonal antibodies directed towards the N-terminal peptide. Samplesinclude: (a) LNCaP cells probed in the presence of N-terminal STEAP-1peptide 1, (b) LNCaP plus non specific peptide 2, (c) normal prostatetissue, (d) grade 3 prostate carcinoma, (e) grade 4 prostate carcinoma,(f) LAPC-9 AD xenograft, (g) normal bladder, (h) normal colon. Allimages are at 400× magnification.

[0031]FIG. 9. Partial nucleotide and deduced amino acid sequences ofSTEAP-2 (98P4B6) clone GTA3 cDNA (SEQ ID NOS: 7 and 8, respectively).The 5′ end sequence of this clone contains an ORF of 173 amino acids.

[0032]FIG. 10. Nucleotide sequences of additional STEAP family membersidentified by searching the dbest database with the protein sequence ofSTEAP-1. In addition to STEAP-1, another three STEAP family members areindicated with their GenBank accession numbers. One of these correspondsto the gene 98P4B6 that was identified by SSH. AA5058880/SEQ ID NO.9;98P4B6 SSH/SEQ ID NO. 10; AI139607/SEQ ID NO. 11; R80991/SEQ ID NO. 12.

[0033]FIG. 11. Primary structural comparison of STEAP family proteins.

[0034]FIG. 11A. Amino acid sequence alignment of STEAP-1 (8P1D4 CLONE10; SEQ ID NO: 1) and STEAP-2 (98P4B6; SEQ ID NO: 7) sequences. Thealignment was performed using the SIM alignment program of the BaylorCollege of Medicine Search Launcher Web site. Results show a 61.4%identity in a 171 amino acid overlap; Score: 576.0; Gap frequency: 0.0%.

[0035]FIG. 11B. Amino acid sequence alignment of STEAP-1 with partialORF sequences of STEAP-2 and two other putative family member proteinsusing PIMA program (PIMA 1.4 program at Internet address<http:\\dot.imgen.bcm.tmc.edu:9331\multi-align\multi-align.html>);transmembrane domains identified by the SOSUI program (available atInternet address <http:\\www.tuat.ac.jp\˜mitaku\adv_sosui\submit.html>).are in bold.

[0036]FIG. 12. Predominant expression of AI139607 in placenta andprostate. First strand cDNA was prepared from 16 normal tissues.Normalization was performed by PCR using primers to actin and GAPDH.Semi-quantitative PCR, using primers to AI139607, shows predominantexpression of AI139607 in placenta and prostate after 25 cycles ofamplification. The following primers were used to amplify AI139607:

[0037] AI139607.1 5′ TTAGGACAACTTGATCACCAGCA 3′ (SEQ ID NO: 13)

[0038] AI139607.2 5′ TGTCCAGTCCAAACTGGGTTATTT3′ (SEQ ID NO: 14)

[0039]FIG. 13. Predominant expression of R80991 in liver. First strandcDNA was prepared from 16 normal tissues. Normalization was performed byPCR using primers to actin and GAPDH. Semi-quantitative PCR, usingprimers to R80991, shows predominant expression of R80991 in liver after25 cycles of amplification. The following primers were used to amplifyR80991:

[0040] R80991.1 5′ AGGGAGTTCAGCTTCGTTCAGTC 3′ (SEQ ID NO: 15)

[0041] R80991.2 5′ GGTAGAACTTGTAGCGGCTCTCCT3′ (SEQ ID NO: 16)

[0042]FIG. 14. Predominant expression of STEAP-2 (98P4B6) in prostatetissue. First strand cDNA was prepared from 8 normal tissues, the LAPCxenografts (4AD, 4AI and 9AD) and HeLa cells. Normalization wasperformed by PCR using primers to actin and GAPDH. Semi-quantitativePCR, using primers to 98P4B6, shows predominant expression of 98P4B6 innormal prostate and the LAPC xenografts. The following primers were usedto amplify STEAP II:

[0043] 98P4B6.1 5′ GACTGAGCTGGAACTGGAATTTGT 3′ (SEQ ID NO: 17)

[0044] 98P4B6.2 5′ TTTGAGGAGACTTCATCTCACTGG 3′ (SEQ ID NO: 18)

[0045]FIG. 15. Lower expression of the prostate-specific STEAP-2/98P4B6gene in prostate cancer xenografts determined by Northern blot analysis.Human normal tissue filters (A and B) were obtained from CLONTECH andcontain 2 μg of mRNA per lane. Xenograft filter (C) was prepared with 10μg of total RNA per lane. The blots were analyzed using the SSH derived98P4B6 clone as probe. All RNA samples were normalized by ethidiumbromide staining.

[0046]FIG. 16. Expression of STEAP-2 in prostate and select cancer celllines as determined by Northern blot analysis. Xenograft and cell linefilters were prepared with 10 μg total RNA per lane. The blots wereanalyzed using an SSH derived 98P4B6 clone as probe. All RNA sampleswere normalized by ethidium bromide staining.

[0047]FIG. 17. Chromosomal localization of STEAP family members. Thechromosomal localizations of the STEAP genes described herein weredetermined using the GeneBridge4 radiation hybrid panel (ResearchGenetics, Huntsville Ala.). The mapping for STEAP-2 and AI139607 wasperformed using the Stanford G3 radiation hybrid panel (ResearchGenetics, Huntsville Ala.).

[0048]FIG. 18. Schematic representation of Intron-Exon boundaries withinthe ORF of human STEAP-1 gene. A total of 3 introns (i) and 4 exons (e)were identified.

[0049]FIG. 19. Zooblot southern analysis of STEAP-1 gene in variousspecies. Genomic DNA was prepared from several different organismsincluding human, monkey, dog, mouse, chicken and Drosophila. Tenmicrograms of each DNA sample was digested with EcoRI, blotted ontonitrocellulose and probed with a STEAP-1 probe. Size standards areindicated on the side in kilobases (kb).

[0050]FIG. 20. Southern blot analysis of mouse BAC with a STEAP-1 probe.DNA was prepared from human cells to isolate genomic DNA and from amouse BAC clone (12P11) that contains the mouse STEAP gene. Each DNAsample was digested with EcoRI, blotted onto nitrocellulose and probed.Eight micrograms of genomic DNA was compared to 250 ng of mouse BAC DNA.

DETAILED DESCRIPTION OF THE INVENTION

[0051] Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

[0052] As used herein, the terms “advanced prostate cancer”, “locallyadvanced prostate cancer”, “advanced disease” and “locally advanceddisease” mean prostate cancers which have extended through the prostatecapsule, and are meant to include stage C disease under the AmericanUrological Association (AUA) system, stage C1-C2 disease under theWhitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM(tumor, node, metastasis) system. In general, surgery is not recommendedfor patients with locally advanced disease, and these patients havesubstantially less favorable outcomes compared to patients havingclinically localized (organ-confined) prostate cancer. Locally advanceddisease is clinically identified by palpable evidence of indurationbeyond the lateral border of the prostate, or asymmetry or indurationabove the prostate base. Locally advanced prostate cancer is presentlydiagnosed pathologically following radical prostatectomy if the tumorinvades or penetrates the prostatic capsule, extends into the surgicalmargin, or invades the seminal vesicles.

[0053] As used herein, the terms “metastatic prostate cancer” and“metastatic disease” mean prostate cancers which have spread to regionallymph nodes or to distant sites, and are meant to include stage Ddisease under the AUA system and stage TxNxM+under the TNM system. As isthe case with locally advanced prostate cancer, surgery is generally notindicated for patients with metastatic disease, and hormonal (androgenablation) therapy is the preferred treatment modality. Patients withmetastatic prostate cancer eventually develop an androgen-refractorystate within 12 to 18 months of treatment initiation, and approximatelyhalf of these patients die within 6 months thereafter. The most commonsite for prostate cancer metastasis is bone. Prostate cancer bonemetastases are, on balance, characteristically osteoblastic rather thanosteolytic (i.e., resulting in net bone formation). Bone metastases arefound most frequently in the spine, followed by the femur, pelvis, ribcage, skull and humerus. Other common sites for metastasis include lymphnodes, lung, liver and brain. Metastatic prostate cancer is typicallydiagnosed by open or laparoscopic pelvic lymphadenectomy, whole bodyradionuclide scans, skeletal radiography, and/or bone lesion biopsy.

[0054] As used herein, the term “polynucleotide” means a polymeric formof nucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

[0055] As used herein, the term “polypeptide” means a polymer of atleast 10 amino acids. Throughout the specification, standard threeletter or single letter designations for amino acids are used.

[0056] As used herein, the terms “hybridize”, “hybridizing”,“hybridizes” and the like, used in the context of polynucleotides, aremeant to refer to conventional hybridization conditions, preferably suchas hybridization in 50% formamide/6× SSC/0.1% SDS/100 μg/ml ssDNA, inwhich temperatures for hybridization are above 37 degrees C. andtemperatures for washing in 0.1× SSC/0.1% SDS are above 55 degrees C.,and most preferably to stringent hybridization conditions.

[0057] In the context of amino acid sequence comparisons, the term“identity” is used to express the percentage of amino acid residues atthe same relative position which are the same. Also in this context, theterm “homology” is used to express the percentage of amino acid residuesat the same relative positions which are either identical or aresimilar, using the conserved amino acid criteria of BLAST analysis, asis generally understood in the art. Further details regarding amino acidsubstitutions, which are considered conservative under such criteria,are provided below.

[0058] Additional definitions are provided throughout the subsectionswhich follow.

[0059] Molecular and biochemical Features of the STEAPs

[0060] The invention relates to a novel family of proteins, termedSTEAPs. Four STEAPs are specifically described herein by way ofstructural, molecular and biochemical features. As is further describedin the Examples which follow, the STEAPs have been characterized in avariety of ways. For example, analyses of nucleotide coding and aminoacid sequences were conducted in order to identify conserved structuralelements within the STEAP family. Extensive RT-PCR and Northern blotanalyses of STEAP mRNA expression were conducted in order to establishthe range of normal and cancerous tissues expressing the various STEAPmessages. Western blot, immunohistochemical and flow cytometric analysesof STEAP protein expression were conducted to determine proteinexpression profiles, cell surface localization and gross moleculartopology of STEAP.

[0061] The prototype member of the STEAP family, STEAP-1, is asix-transmembrane cell surface protein of 339 amino acids with noidentifiable homology to any known human protein. The cDNA nucleotideand deduced amino acid sequences of human STEAP-1 are shown in FIG. 1A.A gross topological schematic of the STEAP-1 protein integrated withinthe cell membrane is shown in FIG. 1B. STEAP-1 expression ispredominantly prostate-specific in normal tissues. Specifically,extensive analysis of STEAP-1 mRNA and protein expression in normalhuman tissues shows that STEAP-1 protein is predominantly expressed inprostate and, to a far smaller degree, in bladder. STEAP-1 mRNA is alsorelatively prostate specific, with only very low level expressiondetected in a few other normal tissues. In cancer, STEAP-1 mRNA andprotein is consistently expressed at high levels in prostate cancer andduring all stages of the disease. STEAP-1 is also expressed in othercancers. Specifically, STEAP-1 mRNA is expressed at very high levels inbladder, colon, pancreatic, and ovarian cancer (as well as othercancers). In addition, cell surface expression of STEAP-1 protein hasbeen established in prostate, bladder and colon cancers. Therefore,STEAP-1 has all of the hallmark characteristics of an excellenttherapeutic target for the treatment of certain cancers, includingparticularly prostate, colon and bladder carcinomas.

[0062] STEAP-2 is a highly homologous transmembrane protein encoded by adistinct gene. The STEAP-1 and STEAP-2 sequences show a high degree ofstructural conservation, particularly throughout their predictedtransmembrane domains. The partial cDNA nucleotide and deduced aminoacid sequences of STEAP-2 are shown in FIG. 9. Both the STEAP-1 andSTEAP-2 genes are located on chromosome 7, but on different arms.STEAP-2 exhibits a markedly different mRNA and protein expressionprofile relative to STEAP-1, suggesting that these two STEAP familymembers may be differentially regulated. STEAP-2 appears to be veryprostate-specific, as significant mRNA expression is not detected in avariety of normal tissues. In prostate cancer, STEAP-2 also appears tofollow a different course relative to STEAP-1, since STEAP-2 expressionis down-regulated in at least some prostate cancers. In addition,STEAP-2 expression in other non-prostate cancers tested seems generallyabsent, although high level expression of STEAP-2 (like STEAP-1) isdetected in Ewing sarcoma.

[0063] STEAP-3 and STEAP-4 appear to be closely related to both STEAP-1and STEAP-2 on a structural level, and both appear to be transmembraneproteins as well. STEAP-3 and STEAP-4 show unique expression profiles.STEAP-3, for example, appears to have an expression pattern which ispredominantly restricted to placenta and, to a smaller degree,expression is seen in prostate but not in other normal tissues tested.STEAP-4 seems to be expressed predominantly in liver. Neither STEAP-3nor STEAP-4 appear to be expressed in prostate cancer xenografts whichexhibit high level STEAP-1 and STEAP-2 expression.

[0064] Three of the four STEAPs described herein map to human chromosome7 (STEAP-1, -2 and 3). Interestingly, STEAP-1 maps within 7p22 (7p22.3),a large region of allelic gain reported for both primary and recurrentprostate cancers (Visakorpi et al., 1995 Cancer Res. 55: 342, Nupponenet al., 1998 American J. Pathol. 153: 141), suggesting thatup-regulation of STEAP-1 in cancer might include genomic mechanisms.

[0065] The function of the STEAPs are not known. Other cell surfacemolecules that contain six transmembrane domains include ion channels(Dolly and Parcej, 1996 J Bioenerg Biomembr 28:231) and water channelsor aquaporins (Reizer et al., 1993 Crit Rev Biochem Mol Biol 28:235).Structural studies show that both types of molecules assemble intotetrameric complexes to form functional channels (Christie, 1995, ClinExp Pharmacol Physiol 22:944, Walz et al., 1997 Nature 387:624, Cheng etal., 1997 Nature 387:627). Immunohistochemical staining of STEAP-1 inthe prostate gland seems to be concentrated at the cell-cell boundaries,with less staining detected at the luminal side. This may suggest a rolefor STEAP-1 in tight-junctions, gap-junctions or cell adhesion. In orderto test these possibilities, xenopus oocytes (or other cells) expressingSTEAP may being analyzed using voltage-clamp and patch-clamp experimentsto determine if STEAP functions as an ion-channel. Oocyte cell volumemay also be measured to determine if STEAP exhibits water channelproperties. If STEAPs function as channel or gap-junction proteins, theymay serve as excellent targets for inhibition using, for example,antibodies, small molecules, and polynucleotides capable of inhibitingexpression or function. The restricted expression pattern In normaltissue, and the high levels of expression in cancer tissue suggest thatinterfering with STEAP function may selectively kill cancer cells.

[0066] Since the STEAP gene family is predominantly expressed inepithelial tissue, it seems possible that the STEAP proteins function asion channels or gap-junction proteins in epithelial cell function. Ionchannels have been implicated in proliferation and invasiveness ofprostate cancer cells (Lalani et al., 1997, Cancer Metastasis Rev16:29). Both rat and human prostate cancer cells contain sub-populationof cells with higher and lower expression levels of sodium channels.Higher levels of sodium channel expression correlate with moreaggressive invasiveness in vitro (Smith et al., 1998, FEBS Lett.423:19). Similarly, it has been shown that a specific blockade of sodiumchannels inhibits the invasiveness of PC-3 cells in vitro (Laniado etal., 1997, Am. J. Pathol. 150:1213), while specific inhibition ofpotassium channels in LNCaP cells inhibited cell proliferation (Skrymaet al., 1997, Prostate 33:112). These reports suggest a role for ionchannels in prostate cancer and also demonstrate that small moleculesthat inhibit ion channel function may interfere with prostate cancerproliferation.

[0067] STEAP Polynucleotides

[0068] One aspect of the invention provides polynucleotidescorresponding or complementary to all or part of a STEAP gene, mRNA,and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding a STEAP protein and fragments thereof, DNA,RNA, DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a STEAP gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides which hybridize toa STEAP gene, mRNA, or to a STEAP-encoding polynucleotide (collectively,“STEAP polynucleotides”). As used herein, STEAP genes and proteins aremeant to include the STEAP-1 and STEAP-2 genes and proteins, the genesand proteins corresponding to GeneBank Accession numbers All 39607 andR80991 (STEAP-3 and STEAP-4, respectively), and the genes and proteinscorresponding to other STEAP proteins and structurally similar variantsof the foregoing. Such other STEAP proteins and variants will generallyhave coding sequences which are highly homologous to the STEAP-1 and/orSTEAP-2 coding sequences, and preferably will share at least about 50%amino acid identity and at least about 60% amino acid homology (usingBLAST criteria), more preferably sharing 70% or greater homology (usingBLAST criteria).

[0069] The STEAP family member gene sequences described herein encodeSTEAP proteins sharing unique highly conserved amino acid sequencedomains which distinguish them from other proteins. Proteins whichinclude one or more of these unique highly conserved domains may berelated to the STEAP family members or may represent new STEAP proteins.Referring to FIG. 11A, which is an amino acid sequence alignment of thefull STEAP-1 and partial STEAP-2 protein sequences, the STEAP-1 andSTEAP-2 sequences share 61% identity and 79% homology, with particularlyclose sequence conservation in the predicted transmembrane domains.Referring to FIG. 11B, which is an amino acid alignment of the availablestructures of the four STEAP family members, very close conservation isapparent in the overlapping regions, particularly in the fourth andfifth transmembrane domains and the predicted intracellular loop betweenthem. Amino acid sequence comparisons show that (1) STEAP-2 and STEAP-3are 50% identical and 69% homologous in their overlapping sequences; (2)STEAP-2 and STEAP-4 are 56% identical and 87% homologous in theiroverlapping sequences; (3) STEAP-3 and STEAP-1 are 37% identical and 63%homologous in their overlapping sequences; (4) STEAP-3 and STEAP-4 are38% identical and 57% homologous in their overlapping sequences; and (5)STEAP 4 and STEAP-1 are 42% identical and 65% homologous in theiroverlapping sequences.

[0070] A STEAP polynucleotide may comprise a polynucleotide having thenucleotide sequence of human STEAP-1 as shown in FIG. 1A (SEQ ID NO. 1)or the nucleotide sequence of human STEAP-2 as shown in FIG. 9 (SEQ IDNO: 7), a sequence complementary to either of the foregoing, or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucelotide which encodes the human STEAP-1 protein aminoacid sequence as shown in FIG. 1A (SEQ ID NO. 2) or which encodes thehuman STEAP-2 protein amino acid sequence as shown in FIG. 9 (SEQ ID NO:8), a sequence complementary to either of the foregoing, or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide which is capable of hybridizing understringent hybridization conditions to the human STEAP-1 cDNA shown inFIG. 1A (SEQ ID NO. 1) or to a polynucleotide fragment thereof. Anotherembodiment comprises a polynucleotide which is capable of hybridizingunder stringent hybridization conditions to the human STEAP-2 cDNA shownin FIG. 9 (SEQ ID NO. 7) or to a polynucleotide fragment thereof.

[0071] Specifically contemplated are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone or including alternative bases, whether derivedfrom natural sources or synthesized. For example, antisense moleculescan be RNAs or other molecules, including peptide nucleic acids (PNAs)or non-nucleic acid molecules such as phosphorothioate derivatives, thatspecifically bind DNA or RNA in a base pair-dependent manner. A skilledartisan can readily obtain these classes of nucleic acid molecules usingthe STEAP polynucleotides and polynucleotide sequences disclosed herein.

[0072] Further specific embodiments of this aspect of the inventioninclude primers and primer pairs, which allow the specific amplificationof the polynucleotides of the invention or of any specific partsthereof, and probes that selectively or specifically hybridize tonucleic acid molecules of the invention or to any part thereof. Probesmay be labeled with a detectable marker, such as, for example, aradioisotope, fluorescent compound, bioluminescent compound, achemiluminescent compound, metal chelator or enzyme. Such probes andprimers can be used to detect the presence of a STEAP polynucleotide ina sample and as a means for detecting a cell expressing a STEAP protein.Examples of such probes include polynucleotides comprising all or partof the human STEAP-1 cDNA sequence shown in FIG. 1A (SEQ ID NO. 1) andpolynucleotides comprising all or part of the human STEAP-2 cDNAsequence shown in FIG. 9 (SEQ ID NO. 7). Examples of primer pairscapable of specifically amplifying STEAP mRNAs are also described in theExamples which follow. As will be understood by the skilled artisan, agreat many different primers and probes may be prepared based on thesequences provided in herein and used effectively to amplify and/ordetect a STEAP mRNA or an mRNA encoding a particular STEAP family member(e.g., STEAP-1).

[0073] As used herein, a polynucleotide is said to be “isolated” when itis substantially separated from contaminant polynucleotides whichcorrespond or are complementary to genes other than the STEAP gene orwhich encode polypeptides other than STEAP gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated STEAP polynucleotide.

[0074] The STEAP polynucleotides of the invention are useful for avariety of purposes, including but not limited to their use as probesand primers for the amplification and/or detection of the STEAP gene(s),mRNA(s), or fragments thereof; as reagents for the diagnosis and/orprognosis of prostate cancer and other cancers; as coding sequencescapable of directing the expression of STEAP polypeptides; as tools formodulating or inhibiting the expression of the STEAP gene(s) and/ortranslation of the STEAP transcript(s); and as therapeutic agents.

[0075] Methods for Isolating STEAP-Encoding Nucleic Acid Molecules

[0076] The STEAP cDNA sequences described herein enable the isolation ofother polynucleotides encoding STEAP gene product(s), as well as theisolation of polynucleotides encoding STEAP gene product homologues,alternatively spliced isoforms, allelic variants, and mutant forms ofthe STEAP gene product. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a STEAP gene are wellknown (See, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition., Cold Spring Harbor Press, New York,1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,Wiley and Sons, 1995). For example, lambda phage cloning methodologiesmay be conveniently employed, using commercially available cloningsystems (e.g., Lambda ZAP Express, Stratagene). Phage clones containingSTEAP gene cDNAs may be identified by probing with a labeled STEAP cDNAor a fragment thereof. For example, in one embodiment, the STEAP-1 cDNA(FIG. 1A) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full length cDNAs corresponding to a STEAPgene. Similarly, the STEAP-2 cDNA sequence may be employed. A STEAP genemay be isolated by screening genomic DNA libraries, bacterial artificialchromosome libraries (BACs), yeast artificial chromosome libraries(YACs), and the like, with STEAP DNA probes or primers.

[0077] Recombinant DNA Molecules and Host-Vector Systems

[0078] The invention also provides recombinant DNA or RNA moleculescontaining a STEAP polynucleotide, including but not limited to phages,plasmids, phagemids, cosmids, YACs, BACs, as well as various viral andnon-viral vectors well known in the art, and cells transformed ortransfected with such recombinant DNA or RNA molecules. As used herein,a recombinant DNA or RNA molecule is a DNA or RNA molecule that has beensubjected to molecular manipulation in vitro. Methods for generatingsuch molecules are well known (see, for example, Sambrook et al, 1989,supra).

[0079] The invention further provides a host-vector system comprising arecombinant DNA molecule containing a STEAP polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 cell). Examples of suitablemammalian cells include various prostate cancer cell lines such LnCaP,PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of a STEAP may be used to generate STEAP proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

[0080] A wide range of host-vector systems suitable for the expressionof STEAP proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, STEAP may be preferably expressed in severalprostate cancer and non-prostate cell lines, including for example 293,293T, rat-1, 3T3, PC-3, LNCaP and TsuPr1. The host-vector systems of theinvention are useful for the production of a STEAP protein or fragmentthereof. Such host-vector systems may be employed to study thefunctional properties of STEAP and STEAP mutations.

[0081] Proteins encoded by the STEAP genes, or by fragments thereof,will have a variety of uses, including but not limited to generatingantibodies and in methods for identifying ligands and other agents andcellular constituents that bind to a STEAP gene product. Antibodiesraised against a STEAP protein or fragment thereof may be useful indiagnostic and prognostic assays, imaging methodologies (including,particularly, cancer imaging), and therapeutic methods in the managementof human cancers characterized by expression of a STEAP protein, such asprostate, colon, breast, cervical and bladder carcinomas, ovariancancers, testicular cancers and pancreatic cancers. Variousimmunological assays useful for the detection of STEAP proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting prostate cells(e.g., in radioscintigraphic imaging methods). STEAP proteins may alsobe particularly useful in generating cancer vaccines, as furtherdescribed below.

[0082] STEAP Proteins

[0083] Another aspect of the present invention provides various STEAPproteins and polypeptide fragments thereof. As used herein, a STEAPprotein refers to a protein that has or includes the amino acid sequenceof human STEAP-1 as provided in FIG. 1A (SEQ ID NO. 2), human STEAP-2 asprovided in FIG. 9 (SEQ ID NO. 8), the amino acid sequence of othermammalian STEAP homologues and variants, as well as allelic variants andconservative substitution mutants of these proteins that have STEAPbiological activity.

[0084] The STEAP proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants and homologs that can be isolated/generated andcharacterized without undue experimentation following the methodsoutlined below. Fusion proteins which combine parts of different STEAPproteins or fragments thereof, as well as fusion proteins of a STEAPprotein and a heterologous polypeptide are also included. Such STEAPproteins will be collectively referred to as the STEAP proteins, theproteins of the invention, or STEAP. As used herein, the term “STEAPpolypeptide” refers to a polypeptide fragment or a STEAP protein of atleast 10 amino acids, preferably at least 15 amino acids.

[0085] A specific embodiment of a STEAP protein comprises a polypeptidehaving the amino acid sequence of human STEAP-1 as shown in FIG. 1A (SEQID NO. 2). Another embodiment of a STEAP protein comprises a polypeptidecontaining the partial STEAP-2 amino acid sequence as shown in FIG. 9(SEQ ID NO. 8). Another embodiment comprises a polypeptide containingthe partial STEAP-3 amino acid sequence of shown in FIG. 11B. Yetanother embodiment comprises a polypeptide containing the partialSTEAP-4 amino acid sequence of shown in FIG. 11B.

[0086] In general, naturally occurring allelic variants of human STEAPwill share a high degree of structural identity and homology (e.g., 90%or more identity). Typically, allelic variants of the STEAP proteinswill contain conservative amino acid substitutions within the STEAPsequences described herein or will contain a substitution of an aminoacid from a corresponding position in a STEAP homologue. One class ofSTEAP allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular STEAP amino acidsequence, but will further contain a radical departure form thesequence, such as a non-conservative substitution, truncation, insertionor frame shift. Such alleles represent mutant STEAP proteins thattypically do not perform the same biological functions or do not haveall of the biological characteristics.

[0087] Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

[0088] STEAP proteins may be embodied in many forms, preferably inisolated form. As used herein, a protein is said to be “isolated” whenphysical, mechanical or chemical methods are employed to remove theSTEAP protein from cellular constituents that are normally associatedwith the protein. A skilled artisan can readily employ standardpurification methods to obtain an isolated STEAP protein. A purifiedSTEAP protein molecule will be substantially free of other proteins ormolecules which impair the binding of STEAP to antibody or other ligand.The nature and degree of isolation and purification will depend on theintended use. Embodiments of a STEAP protein include a purified STEAPprotein and a functional, soluble STEAP protein. In one form, suchfunctional, soluble STEAP proteins or fragments thereof retain theability to bind antibody or other ligand.

[0089] The invention also provides STEAP polypeptides comprisingbiologically active fragments of the STEAP amino acid sequence, such asa polypeptide corresponding to part of the amino acid sequences forSTEAP-1 as shown in FIG. 1A (SEQ ID NO. 2), STEAP-2 as shown in FIG. 9(SEQ ID NO: 8), or STEAP-3 or STEAP-4 as shown in FIG. 11B. Suchpolypeptides of the invention exhibit properties of a STEAP protein,such as the ability to elicit the generation of antibodies whichspecifically bind an epitope associated with a STEAP protein.Polypeptides comprising amino acid sequences which are unique to aparticular STEAP protein (relative to other STEAP proteins) may be usedto generate antibodies which will specifically react with thatparticular STEAP protein. For example, referring to the amino acidalignment of the STEAP-1 and STEAP-2 structures shown in FIG. 11A, theskilled artisan will readily appreciate that each molecule containsstretches of sequence unique to its structure. These unique stretchescan be used to generate STEAP-1 or STEAP-2 specific antibodies.

[0090] STEAP polypeptides can be generated using standard peptidesynthesis technology or using chemical cleavage methods well known inthe art based on the amino acid sequences of the human STEAP proteinsdisclosed herein. Alternatively, recombinant methods can be used togenerate nucleic acid molecules that encode a polypeptide fragment of aSTEAP protein. In this regard, the STEAP-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of STEAPproteins. STEAP polypeptides are particularly useful in generating andcharacterizing domain specific antibodies (e.g., antibodies recognizingan extracellular or intracellular epitope of a STEAP protein), ingenerating STEAP family member specific antibodies (e.g., anti-STEAP-1,anti-STEAP 2 antibodies), identifying agents or cellular factors thatbind to a particular STEAP or STEAP domain, and in various therapeuticcontexts, including but not limited to cancer vaccines. STEAPpolypeptides containing particularly interesting structures can bepredicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-STEAP antibodies or in identifying cellular factors thatbind to STEAP.

[0091] STEAP Antibodies

[0092] Another aspect of the invention provides antibodies that bind toSTEAP proteins and polypeptides. The most preferred antibodies willselectively bind to a STEAP protein and will not bind (or will bindweakly) to non-STEAP proteins and polypeptides. Anti-STEAP antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule whichbinds to its target, i.e., the antigen binding region.

[0093] For some applications, it may be desirable to generate antibodieswhich specifically react with a particular STEAP protein and/or anepitope within a particular structural domain. For example, preferredantibodies useful for cancer therapy and diagnostic imaging purposes arethose which react with an epitope in an extracellular region of theSTEAP protein as expressed in cancer cells. Such antibodies may begenerated by using the STEAP proteins described herein, or usingpeptides derived from predicted extracellular domains thereof, as animmunogen. In this regard, with reference to the STEAP-1 proteintopological schematic shown in FIG. 1B, regions in the extracellularloops between the indicated transmembrane domains may be selected asused to design appropriate immunogens for raising extracellular-specificantibodies.

[0094] STEAP antibodies of the invention may be particularly useful inprostate cancer therapeutic strategies, diagnostic and prognosticassays, and imaging methodologies. The invention provides variousimmunological assays useful for the detection and quantification ofSTEAP and mutant STEAP proteins and polypeptides. Such assays generallycomprise one or more STEAP antibodies capable of recognizing and bindinga STEAP or mutant STEAP protein, as appropriate, and may be performedwithin various immunological assay formats well known in the art,including but not limited to various types of radioimmunoassays,enzyme-linked immunosorbent assays (ELISA), enzyme-linkedimmunofluorescent assays (ELIFA), and the like. In addition,immunological imaging methods capable of detecting prostate cancer arealso provided by the invention, including but limited toradioscintigraphic imaging methods using labeled STEAP antibodies. Suchassays may be clinically useful in the detection, monitoring, andprognosis of prostate cancer, particularly advanced prostate cancer.

[0095] STEAP antibodies may also be used in methods for purifying STEAPand mutant STEAP proteins and polypeptides and for isolating STEAPhomologues and related molecules. For example, in one embodiment, themethod of purifying a STEAP protein comprises incubating a STEAPantibody, which has been coupled to a solid matrix, with a lysate orother solution containing STEAP under conditions which permit the STEAPantibody to bind to STEAP; washing the solid matrix to eliminateimpurities; and eluting the STEAP from the coupled antibody. Other usesof the STEAP antibodies of the invention Include generatinganti-idiotypic antibodies that mimic the STEAP protein.

[0096] STEAP antibodies may also be used therapeutically by, forexample, modulating or inhibiting the biological activity of a STEAPprotein or targeting and destroying prostate cancer cells expressing aSTEAP protein. Antibody therapy of prostate and other cancers is morespecifically described in a separate subsection below.

[0097] Various methods for the preparation of antibodies are well knownin the art. For example, antibodies may be prepared by immunizing asuitable mammalian host using a STEAP protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of STEAP may alsobe used, such as a STEAP GST-fusion protein. In a particular embodiment,a GST fusion protein comprising all or most of the open reading frameamino acid sequence of FIG. 1A may be produced and used as an immunogento generate appropriate antibodies. Cells expressing or overexpressingSTEAP may also be used for immunizations. Similarly, any cell engineeredto express STEAP may be used. Such strategies may result in theproduction of monoclonal antibodies with enhanced capacities forrecognizing endogenous STEAP. Another useful immunogen comprises STEAPproteins linked to the plasma membrane of sheep red blood cells.

[0098] The amino acid sequence of STEAP as shown in FIG. 1A (SEQ ID NO.2) may be used to select specific regions of the STEAP protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the STEAP amino acid sequence may be used to identifyhydrophilic regions in the STEAP structure. Regions of the STEAP proteinthat show immunogenic structure, as well as other regions and domains,can readily be identified using various other methods known in the art,such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. For the generation ofantibodies which specifically recognize a mutant STEAP protein, aminoacid sequences unique to the mutant (relative to wild type STEAP) arepreferable.

[0099] Methods for preparing a protein or polypeptide for use as animmunogen and for preparing immunogenic conjugates of a protein with acarrier such as BSA, KLH, or other carrier proteins are well known inthe art. In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a STEAP immunogen is conducted generally byinjection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

[0100] STEAP monoclonal antibodies are preferred and may be produced byvarious means well known in the art. For example, immortalized celllines which secrete a desired monoclonal antibody may be prepared usingthe standard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the STEAP protein orSTEAP fragment. When the appropriate Immortalized cell culture secretingthe desired antibody is identified, the cells may be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

[0101] As mentioned above, numerous STEAP polypeptides may be used asimmunogens for generating monoclonal antibodies using traditionalmethods. A particular embodiment comprises an antibody whichimmunohistochemically stains 293T cells transfected with an expressionplasmid carrying the STEAP-1 coding sequence, the transfected cellsexpressing STEAP-1 protein, but does immunohistochemically stainuntransfected 293T cells. An assay for characterizing such antibodies isprovided in Example 5 herein.

[0102] In another embodiment, STEAP-1 monoclonal antibodies may begenerated using NIH 3T3 cells expressing STEAP-1 as an immunogen togenerate mAbs that recognize the cell surface epitopes of STEAP-1.Reactive mAbs may be screened by cell-based ELISAs using PC-3 cellsover-expressing STEAP-1. In another specific embodiment, 3 peptidesrepresenting the extracellular regions of the STEAP-1 protein(specifically, REVIHPLATSHQQYFYKIPILV (SEQ ID NO. 19),RRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEI (SEQ ID NO. 20) and WIDIKQFVWYTPPTF(SEQ ID NO. 21) are coupled to sheep red blood cells for immunization.In another specific embodiment, recombinant STEAP-1 protein generatedwith an amino-terminal His-tag using a suitable expression system (e.g.,baculovirus expression system pBlueBac4.5, Invitrogen) is purified usinga Nickel column and used as immunogen.

[0103] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the STEAP protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.Humanized or human STEAP antibodies may also be produced and arepreferred for use in therapeutic contexts. Various approaches forproducing such humanized antibodies are known, and include chimeric andCDR grafting methods; methods for producing fully human monoclonalantibodies include phage display and transgenic methods (for review, seeVaughan et al., 1998, Nature Biotechnology 16: 535-539).

[0104] Fully human STEAP monoclonal antibodies may be generated usingcloning technologies employing large human Ig gene combinatoriallibraries (i.e., phage display) (Griffiths and Hoogenboom, Building anin vitro immune system: human antibodies from phage display libraries.In: Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human STEAP monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

[0105] Reactivity of STEAP antibodies with a STEAP protein may beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,STEAP proteins, peptides, STEAP-expressing cells or extracts thereof.

[0106] A STEAP antibody or fragment thereof of the invention may belabeled with a detectable marker or conjugated to a second molecule,such as a cytotoxic agent, and used for targeting the second molecule toa STEAP positive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy,in DeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice ofOncology, 4th ed., J. B. Lippincott Co., Philadelphia, 2624-2636).Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme.

[0107] Methods for the Detection of STEAP

[0108] Another aspect of the present invention relates to methods fordetecting STEAP polynucleotides and STEAP proteins, as well as methodsfor identifying a cell which expresses STEAP.

[0109] More particularly, the invention provides assays for thedetection of STEAP polynucleotides in a biological sample, such asserum, bone, prostate, and other tissues, urine, semen, cellpreparations, and the like. Detectable STEAP polynucleotides include,for example, a STEAP gene or fragments thereof, STEAP mRNA, alternativesplice variant STEAP mRNAs, and recombinant DNA or RNA moleculescontaining a STEAP polynucleotide. A number of methods for amplifyingand/or detecting the presence of STEAP polynucleotides are well known inthe art and may be employed in the practice of this aspect of theinvention.

[0110] In one embodiment, a method for detecting a STEAP mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing a STEAP polynucleotides as sense and antisense primers to amplifySTEAP cDNAs therein; and detecting the presence of the amplified STEAPcDNA. In another embodiment, a method of detecting a STEAP gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using STEAP polynucleotides as senseand antisense primers to amplify the STEAP gene therein; and detectingthe presence of the amplified STEAP gene. Any number of appropriatesense and antisense probe combinations may be designed from thenucleotide sequences provided for STEAP-1 (FIG. 1A; SEQ ID NO. 1),STEAP-2 (FIG. 9; SEQ ID NO. 7), STEAP-3 (FIG. 10; SEQ ID NO. 11), orSTEAP-4 (FIG. 10; SEQ ID NO. 12), as appropriate, and used for thispurpose.

[0111] The invention also provides assays for detecting the presence ofa STEAP protein in a tissue of other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a STEAP protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western Blot analysis, molecular binding assays, ELISA, ELIFA and thelike.

[0112] For example, in one embodiment, a method of detecting thepresence of a STEAP protein in a biological sample comprises firstcontacting the sample with a STEAP antibody, a STEAP-reactive fragmentthereof, or a recombinant protein containing an antigen binding regionof a STEAP antibody; and then detecting the binding of STEAP protein inthe sample thereto.

[0113] Methods for identifying a cell which expresses STEAP are alsoprovided. In one embodiment, an assay for identifying a cell whichexpresses a STEAP gene comprises detecting the presence of STEAP mRNA inthe cell. Methods for the detection of particular mRNAs in cells arewell known and include, for example, hybridization assays usingcomplementary DNA probes (such as in situ hybridization using labeledSTEAP riboprobes, Northern blot and related techniques) and variousnucleic acid amplification assays (such as RT-PCR using complementaryprimers specific for STEAP, and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell which expresses a STEAPgene comprises detecting the presence of STEAP protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and may be employed for the detection of STEAPproteins and STEAP expressing cells.

[0114] STEAP expression analysis may also be useful as a tool forIdentifying and evaluating agents which modulate STEAP gene expression.For example, STEAP-1 expression is significantly upregulated in colon,bladder, pancreatic, ovarian and other cancers. Identification of amolecule or biological agent that could inhibit STEAP-1 over-expressionmay be of therapeutic value in the treatment of cancer. Such an agentmay be identified by using a screen that quantifies STEAP expression byRT-PCR, nucleic acid hybridization or antibody binding.

[0115] Assays for Determining STEAP Expression Status

[0116] Determining the status of STEAP expression patterns in anindividual may be used to diagnose cancer and may provide prognosticinformation useful in defining appropriate therapeutic options.Similarly, the expression status of STEAP may provide information usefulfor predicting susceptibility to particular disease stages, progression,and/or tumor aggressiveness. The invention provides methods and assaysfor determining STEAP expression status and diagnosing cancers whichexpress STEAP.

[0117] In one aspect, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant increase in STEAP mRNA or protein expression ina test cell or tissue sample relative to expression levels in thecorresponding normal cell or tissue. In one embodiment, the presence ofSTEAP-1 mRNA is evaluated in tissue samples of the colon, pancreas,bladder, ovary, cervix, testis or breast. The presence of significantSTEAP-1 expression in any of these tissues may be useful to indicate theemergence, presence and/or severity of these cancers, since thecorresponding normal tissues do not express STEAP-1 mRNA. In a relatedembodiment, STEAP-1 expression status may be determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodor assay would comprise determining the level of STEAP-1 proteinexpressed by cells in a test tissue sample and comparing the level sodetermined to the level of STEAP expressed in a corresponding normalsample. In one embodiment, the presence of STEAP-1 protein is evaluated,for example, using immunohistochemical methods. STEAP antibodies orbinding partners capable of detecting STEAP protein expression may beused in a variety of assay formats well known in the art for thispurpose.

[0118] Peripheral blood may be conveniently assayed for the presence ofcancer cells, including prostate, colon, pancreatic, bladder and ovariancancers, using RT-PCR to detect STEAP-1 expression. The presence ofRT-PCR amplifiable STEAP-1 mRNA provides an indication of the presenceof one of these types of cancer. RT-PCR detection assays for tumor cellsin peripheral blood are currently being evaluated for use in thediagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston etal., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays are well known inthe art.

[0119] In another approach, a recently described sensitive assay fordetecting and characterizing carcinoma cells in blood may be used(Racila et al., 1998, Proc. Natl. Acad. Sci. USA 95: 4589-4594). Thisassay combines immunomagnetic enrichment with multiparameter flowcytometric and immunohistochemical analyses, and is highly sensitive forthe detection of cancer cells in blood, reportedly capable of detectingone epithelial cell in 1 ml of peripheral blood.

[0120] A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detectingSTEAP mRNA or STEAP protein in a tissue sample, its presence indicatingsusceptibility to cancer, wherein the degree of STEAP mRNA expressionpresent is proportional to the degree of susceptibility.

[0121] Yet another related aspect of the invention is directed tomethods for gauging tumor aggressiveness. In one embodiment, a methodfor gauging aggressiveness of a tumor comprises determining the level ofSTEAP mRNA or STEAP protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of STEAP mRNA or STEAPprotein expressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree ofSTEAP mRNA or STEAP protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness.

[0122] Methods for detecting and quantifying the expression of STEAPmRNA or protein are described herein and use standard nucleic acid andprotein detection and quantification technologies well known in the art.Standard methods for the detection and quantification of STEAP mRNAinclude in situ hybridization using labeled STEAP riboprobes, Northernblot and related techniques using STEAP polynucleotide probes, RT-PCRanalysis using primers specific for STEAP, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RT-PCR may be usedto detect and quantify STEAP mRNA expression as described in theExamples which follow. Any number of primers capable of amplifying STEAPmay be used for this purpose, including but not limited to the variousprimer sets specifically described herein. Standard methods for thedetection and quantification of protein may be used for this purpose. Ina specific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the wild-type STEAP protein may be used in animmunohistochemical assay of biopsied tissue.

[0123] Diagnostic Imaging of Human Cancers

[0124] The expression profiles of STEAP-1 and STEAP-2 indicateantibodies specific therefor may be particularly useful in radionuclideand other forms of diagnostic imaging of certain cancers. For example,immunohistochemical analysis of STEAP-1 protein suggests that in normaltissues STEAP-1 is predominantly restricted to prostate and bladder. Thetransmembrane orientation of STEAP-1 (and presumably STEAP-2) provides atarget readily identifiable by antibodies specifically reactive withextracellular epitopes. This tissue restricted expression, and thelocalization of STEAP to the cell surface of multiple cancers makesSTEAP an ideal candidate for diagnostic imaging. Accordingly, in vivoimaging techniques may be used to image human cancers expressing a STEAPprotein.

[0125] For example, cell surface STEAP-1 protein is expressed at veryhigh levels in several human cancers, particularly prostate, bladder,colon and ovarian cancers, and Ewing sarcoma. Moreover, in normaltissues, STEAP-1 protein expression is largely restricted to prostate.Thus, radiolabeled antibodies specifically reactive with extracellularepitopes of STEAP-1 may be particularly useful in in vivo imaging ofsolid tumors of the foregoing cancers. Such labeled anti-STEAP-1antibodies may provide very high level sensitivities for the detectionof metastasis of these cancers.

[0126] Preferably, monoclonal antibodies are used in the diagnosticimaging methods of the invention.

[0127] Cancer Immunotherapy and Cancer Vaccines

[0128] The invention provides various immunotherapeutic methods fortreating prostate cancer, including antibody therapy, in vivo vaccines,and ex vivo immunotherapy methods, which utilize polynucleotides andpolypeptides corresponding to STEAP and STEAP antibodies. Thesetherapeutic applications are described further in the followingsubsections.

[0129] Applicants have accumulated strong and compelling evidence thatSTEAP-1 is strongly expressed uniformly over the surface of glandularepithelial cells within prostate and prostate cancer cells. See, fordetails, immunohistochemical and Western blot analyses of STEAP-1protein expression presented in Examples 3C and 3D as well as theSTEAP-1 mRNA expression profiles obtained from the Northern blot andRT-PCR generated data presented in Examples 1 and 3A,B. In particular,immunohistochemical analysis results show that the surface of humanprostate epithelial cells (normal and cancer) appear to be uniformlycoated with STEAP-1. Biochemical analysis confirms the cell surfacelocalization of STEAP-1 initially suggested by its putative6-transmembrane primary structural elements and by the pericellularstaining plainly visualized by immunohistochemical staining.

[0130] STEAP-1 is uniformly expressed at high levels over the surface ofprostate glandular epithelia, an ideal situation for immunotherapeuticintervention strategies which target extracellular STEAP epitopes.Systemic administration of STEAP-immunoreactive compositions would beexpected to result in extensive contact of the composition with prostateepithelial cells via binding to STEAP-1 extracellular epitopes.Moreover, given the near absence of STEAP-1 protein expression in normalhuman tissues, there is ample reason to expect exquisite sensitivitywithout toxic, non-specific and/or non-target effects caused by thebinding of the immunotherapeutic composition to STEAP-1 on non-targetorgans and tissues.

[0131] In addition to the high level expression of STEAP-1 in prostateand prostate cancer cells, STEAP-1 appears to be substantiallyover-expressed in a variety of other human cancers, including bladder,colon, pancreatic and ovarian cancers. In particular, high level STEAP-1mRNA expression is detected in all tested prostate cancer tissues andcell lines, and in most of the pancreatic, colon, and bladder cancercell lines tested. High level expression of STEAP-1 is also observed insome ovarian cancer cell lines. Lower level expression is observed insome breast, testicular, and cervical cancer cell lines. Very high levelexpression is also detected in a Ewing sarcoma cell line. Applicantshave shown that cell surface STEAP-1 protein is expressed in bladder andcolon cancers, while there is no detectable cell surface (orintracellular) STEAP-1 protein in normal colon and low expression innormal bladder. Antibodies specifically reactive with extracellulardomains of STEAP-1 may be useful to treat these cancers systemically,either as toxin or therapeutic agent conjugates or as naked antibodiescapable of inhibiting cell proliferation or function.

[0132] STEAP-2 protein is also expressed in prostate cancer, and may beexpressed in other cancers as well. STEAP-2 mRNA analysis by RT-PCR andNorthern blot show that expression is restricted to prostate in normaltissues, is also expressed in some prostate, pancreatic, colon,testicular, ovarian and other cancers. Therefore, antibodies reactivewith STEAP-2 may be useful in the treatment of prostate and othercancers. Similarly, although not yet characterized by applicants, theexpression of STEAP-3 and STEAP-4 (as well as other STEAPs) may beassociated with some cancers. Thus antibodies reactive with these STEAPfamily member proteins may also be useful therapeutically.

[0133] STEAP antibodies may be introduced into a patient such that theantibody binds to STEAP on the cancer cells and mediates the destructionof the cells and the tumor and/or inhibits the growth of the cells orthe tumor. Mechanisms by which such antibodies exert a therapeuticeffect may include complement-mediated cytolysis, antibody-dependentcellular cytotoxicity, modulating the physiologic function of STEAP,inhibiting ligand binding or signal transduction pathways, modulatingtumor cell differentiation, altering tumor angiogenesis factor profiles,and/or by inducing apoptosis. STEAP antibodies conjugated to toxic ortherapeutic agents may also be used therapeutically to deliver the toxicor therapeutic agent directly to STEAP-bearing tumor cells.

[0134] Cancer immunotherapy using anti-STEAP antibodies may follow theteachings generated from various approaches which have been successfullyemployed with respect to other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit Rev Immunol 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186;Tsunenari et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyket al., 1992, Cancer Res 52: 2771-2776), B-cell lymphoma (Funakoshi etal., 1996, J Immunther Emphasis Tumor Immunol 19: 93-101), leukemia(Zhong et al., 1996, Leuk Res 20: 581-589), colorectal cancer (Moun etal., 1994, Cancer Res 54: 6160-6166); Velders et al., 1995, Cancer Res55: 4398-4403), and breast cancer (Shepard et al., 1991, J Clin Immunol11: 117-127).

[0135] Although STEAP antibody therapy may be useful for all stages ofthe foregoing cancers, antibody therapy may be particularly appropriateand in advanced or metastatic cancers. Combining the antibody therapymethod of the invention with a chemotherapeutic or radiation regimen maybe preferred in patients who have not received chemotherapeutictreatment, whereas treatment with the antibody therapy of the inventionmay be indicated for patients who have received one or morechemotherapy. Additionally, antibody therapy may also enable the use ofreduced dosages of concomitant chemotherapy, particularly in patientsthat do not tolerate the toxicity of the chemotherapeutic agent verywell.

[0136] It may be desirable for non-prostate cancer patients to beevaluated for the presence and level of STEAP over-expression,preferably using immunohistochemical assessments of tumor tissue,quantitative STEAP imaging, or other techniques capable of reliablyindicating the presence and degree of STEAP overexpression.Immunohistochemical analysis of tumor biopsies or surgical specimens maybe preferred for this purpose. Methods for immunohistochemical analysisof tumor tissues are well known in the art.

[0137] Anti-STEAP monoclonal antibodies useful in treating prostate andother cancers include those which are capable of initiating a potentimmune response against the tumor and those which are capable of directcytotoxicity. In this regard, anti-STEAP mAbs may elicit tumor celllysis by either complement-mediated or antibody-dependent cellcytotoxicity (ADCC) mechanisms, both of which require an intact Fcportion of the immunoglobulin molecule for interaction with effectorcell Fc receptor sites or complement proteins. In addition, anti-STEAPmAbs which exert a direct biological effect on tumor growth are usefulin the practice of the invention. Potential mechanisms by which suchdirectly cytotoxic mAbs may act include inhibition of cell growth,modulation of cellular differentiation, modulation of tumor angiogenesisfactor profiles, and the induction of apoptosis. The mechanism by whicha particular anti-STEAP mAb exerts an anti-tumor effect may be evaluatedusing any number of in vitro assays designed to determine ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

[0138] The anti-tumor activity of a particular anti-STEAP mAb, orcombination of anti-STEAP mAbs, may be evaluated in vivo using asuitable animal model. For example, xenogenic prostate cancer modelswherein human prostate cancer explants or passaged xenograft tissues areintroduced into immune compromised animals, such as nude or SCID mice,are appropriate in relation to prostate cancer and have been described(Klein et al., 1997, Nature Medicine 3: 402-408). For Example, PCTPatent Application WO98/16628, Sawyers et al., published Apr. 23, 1998,describes various xenograft models of human prostate cancer capable ofrecapitulating the development of primary tumors, micrometastasis, andthe formation of osteoblastic metastases characteristic of late stagedisease. Efficacy may be predicted using assays which measure inhibitionof tumor formation, tumor regression or metastasis, and the like.

[0139] It should be noted that the use of murine or other non-humanmonoclonal antibodies, human/mouse chimeric mAbs may induce moderate tostrong immune responses in some patients. In the most severe cases, suchan immune response may lead to the extensive formation of immunecomplexes which, potentially, can cause renal failure. Accordingly,preferred monoclonal antibodies used in the practice of the therapeuticmethods of the invention are those which are either fully human orhumanized and which bind specifically to the target 20P1F12/TMPRSS2antigen with high affinity but exhibit low or no antigenicity in thepatient.

[0140] The method of the invention contemplate the administration ofsingle anti-STEAP mAbs as well as combinations, or “cocktails, ofdifferent mAbs. Such mAb cocktails may have certain advantages inasmuchas they contain mAbs which exploit different effector mechanisms orcombine directly cytotoxic mAbs with mAbs that rely on immune effectorfunctionality. Such mAbs in combination may exhibit synergistictherapeutic effects. In addition, the administration of anti-STEAP mAbsmay be combined with other therapeutic agents, including but not limitedto various chemotherapeutic agents, androgen-blockers, and immunemodulators (e.g., IL-2, GM-CSF). The anti-STEAP mAbs may be administeredin their “naked” or unconjugated form, or may have therapeutic agentsconjugated to them.

[0141] The anti-STEAP monoclonal antibodies used in the practice of themethod of the invention may be formulated into pharmaceuticalcompositions comprising a carrier suitable for the desired deliverymethod. Suitable carriers include any material which when combined withthe anti-STEAP mAbs retains the anti-tumor function of the antibody andis non-reactive with the subject's immune systems. Examples Include, butare not limited to, any of a number of standard pharmaceutical carrierssuch as sterile phosphate buffered saline solutions, bacteriostaticwater, and the like.

[0142] The anti-STEAP antibody formulations may be administered via anyroute capable of delivering the antibodies to the tumor site.Potentially effective routes of administration include, but are notlimited to, intravenous, intraperitoneal, intramuscular, intratumor,intradermal, and the like. The preferred route of administration is byintravenous injection. A preferred formulation for intravenous injectioncomprises the anti-STEAP mAbs in a solution of preserved bacteriostaticwater, sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. The anti-STEAP mAb preparation may be lyophilized and stored as asterile powder, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

[0143] Treatment will generally involve the repeated administration ofthe anti-STEAP antibody preparation via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. Doses in therange of 10-500 mg mAb per week may be effective and well tolerated.Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti- STEAP mAb preparation may represent an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the mAb or mAbs used, the degree of STEAPoverexpression in the patient, the extent of circulating shed STEAPantigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

[0144] Optimally, patients should be evaluated for the level ofcirculating shed STEAP antigen in serum in order to assist in thedetermination of the most effective dosing regimen and related factors.Such evaluations may also be used for monitoring purposes throughouttherapy, and may be useful to gauge therapeutic success in combinationwith evaluating other parameters (such as serum PSA levels in prostatecancer therapy).

[0145] Cancer Vaccines

[0146] The invention further provides prostate cancer vaccinescomprising a STEAP protein or fragment thereof. The use of a tumorantigen in a vaccine for generating humoral and cell-mediated immunityfor use in anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63: 231-237; Fong et al., 1997, J.Immunol. 159: 3113-3117). Such methods can be readily practiced byemploying a STEAP protein, or fragment thereof, or a STEAP-encodingnucleic acid molecule and recombinant vectors capable of expressing andappropriately presenting the STEAP immunogen.

[0147] For example, viral gene delivery systems may be used to deliver aSTEAP-encoding nucleic acid molecule. Various viral gene deliverysystems which can be used in the practice of this aspect of theinvention include, but are not limited to, vaccinia, fowipox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a STEAP protein or fragment thereof introduced into the patient(e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human STEAP cDNA may be employed. In anotherembodiment, STEAP nucleic acid molecules encoding specific cytotoxic Tlymphocyte (CTL) epitopes may be employed. CTL epitopes can bedetermined using specific algorithms (e.g., Epimer, Brown University) toidentify peptides within a STEAP protein which are capable of optimallybinding to specified HLA alleles.

[0148] Various ex vivo strategies may also be employed. One approachinvolves the use of dendritic cells to present STEAP antigen to apatient's immune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to presentSTEAP peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed withSTEAP peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete STEAP protein.Yet another embodiment involves engineering the overexpression of theSTEAP gene in dendritic cells using various implementing vectors knownin the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther.4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribaset al., 1997, Cancer Res. 57: 2865-2869), and tumor-derived RNAtransfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182).

[0149] Anti-idiotypic anti-STEAP antibodies can also be used inanti-cancer therapy as a vaccine for inducing an immune response tocells expressing a STEAP protein. Specifically, the generation ofanti-idiotypic antibodies is well known in the art and can readily beadapted to generate anti-idiotypic anti-STEAP antibodies that mimic anepitope on a STEAP protein (see, for example, Wagner et al., 1997,Hybridoma 16: 33-40; Foon et al., 1995, J Clin Invest 96: 334-342;Herlyn et al., 1996, Cancer Immunol Immunother 43: 65-76). Such ananti-idiotypic antibody can be used in anti-idiotypic therapy aspresently practiced with other anti-idiotypic antibodies directedagainst tumor antigens.

[0150] Genetic immunization methods may be employed to generateprophylactic or therapeutic humoral and cellular immune responsesdirected against cancer cells expressing STEAP. Constructs comprisingDNA encoding a STEAP protein/immunogen and appropriate regulatorysequences may be injected directly into muscle or skin of an individual,such that the cells of the muscle or skin take-up the construct andexpress the encoded STEAP protein/immunogen. Expression of the STEAPprotein immunogen results in the generation of prophylactic ortherapeutic humoral and cellular immunity against prostate cancer.Various prophylactic and therapeutic genetic immunization techniquesknown in the art may be used (for review, see information and referencespublished at Internet address www.genweb.com).

[0151] Kits

[0152] For use in the diagnostic and therapeutic applications describedor suggested above, kits are also provided by the invention. Such kitsmay comprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe which is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a STEAP proteinor a STEAP gene or message, respectively. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradionucleotide label.

EXAMPLES

[0153] Various aspects of the invention are further described andillustrated by way of the several examples which follow, none of whichare intended to limit the scope of the invention.

Example 1 Isolation of cDNA Fragment of STEAP-1 Gene

[0154] Materials and Methods

[0155] Cell Lines and Human Tissues

[0156] All human cancer cell lines used in this study were obtained fromthe ATCC. All cell lines were maintained in DMEM with 10% fetal calfserum. PrEC (primary prostate epithelial cells) were obtained fromClonetics and were grown in PrEBM media supplemented with growth factors(Clonetics).

[0157] All human prostate cancer xenografts were originally provided byCharles Sawyers (UCLA) (Klein et al., 1997). LAPC-4 AD and LAPC-9 ADxenografts were routinely passaged as small tissue chunks in recipientSCID males. LAPC-4 AI and LAPC-9 AI xenografts were derived as describedpreviously (Klein et al., 1997) and were passaged in castrated males orin female SCID mice. A benign prostatic hyperplasia tissue sample waspatient-derived.

[0158] Human tissues for RNA and protein analyses were obtained from theHuman Tissue Resource Center (HTRC) at the UCLA (Los Angeles, Calif.)and from QualTek, Inc. (Santa Barbara, Calif.).

[0159] RNA Isolation:

[0160] Tumor tissue and cell lines were homogenized in Trizol reagent(Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cellsto isolate total RNA. Poly A RNA was purified from total RNA usingQiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA werequantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzedby gel electrophoresis.

[0161] Oligonucleotides:

[0162] The following HPLC purified oligonucleotides were used. RSACDN(cDNA synthesis primer): 5′TTTTGTACAAGCTT₃₀3′ (SEQ ID NO.22) Adaptor 1:5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT3′ (SEQ ID NO.23)                            3′GGCCCGTCCA5′ Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT3′ (SEQ ID NO.24)                            3′CGGCTCCA5′ PCR primer 1:5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO.25) Nested primer (NP)1:5′TCGAGCGGCCGCCCGGGCAGGT3′ (SEQ ID NO.26) Nested primer (NP)2:5′AGCGTGGTCGCGGCCGAGGT3′ (SEQ ID NO.27

[0163] Suppression Subtractive Hybridization:

[0164] Suppression Subtractive Hybridization (SSH) was used to identifycDNAs corresponding to genes which may be up-regulated in androgendependent prostate cancer compared to benign prostatic hyperplasia.

[0165] Double stranded cDNAs corresponding to the LAPC-4 AD xenograft(tester) and the BPH tissue (driver) were synthesized from 2 μg ofpoly(A)⁺ RNA isolated from xenograft and BPH tissue, as described above,using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng ofoligonucleotide RSACDN as primer. First- and second-strand synthesiswere carried out as described in the Kit's user manual protocol(CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resultingcDNA was digested with Rsa I for 3 hrs. at 37° C. Digested cDNA wasextracted with phenol/chloroform (1:1) and ethanol precipitated.

[0166] Driver cDNA (BPH) was generated by combining in a 4 to 1 ratioRsa I digested BPH cDNA with digested cDNA from mouse liver, in order toensure that murine genes were subtracted from the tester cDNA (LAPC-4AD).

[0167] Tester cDNA (LAPC-4 AD) was generated by diluting 1 μl of Rsa Idigested LAPC-4 AD cDNA (400 ng) in 5 μl of water. The diluted cDNA (2μl, 160 ng) was then ligated to 2 μl of adaptor 1 and adaptor 2 (10 μM),in separate ligation reactions, in a total volume of 10 μl at 16° C.overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

[0168] The first hybridization was performed by adding 1.5 μl (600 ng)of driver cDNA to each of two tubes containing 1.5 μl (20 ng) adaptor 1-and adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlayed with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

[0169] PCR Amplification, Cloning and Sequencing of Gene FragmentsGenerated from SSH:

[0170] To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10uM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

[0171] The PCR products were inserted into pCR2.1 using the T/A vectorcloning kit (Invitrogen). Transformed E. coli were subjected toblue/white and ampicillin selection. White colonies were picked andarrayed into 96 well plates and were grown in liquid culture overnight.To identify inserts, PCR amplification was performed on 1 ml ofbacterial culture using the conditions of PCR1 and NP1 and NP2 asprimers. PCR products were analyzed using 2% agarose gelelectrophoresis.

[0172] Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

[0173] RT-PCR Expression Analysis:

[0174] First strand cDNAs were generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturers protocol was used and included an incubationfor 50 min at 42° C. with reverse transcriptase followed by RNAse Htreatment at 37° C. for 20 min. After completing the reaction, thevolume was increased to 200 μl with water prior to normalization. Firststrand cDNAs from 16 different normal human tissues were obtained fromClontech.

[0175] Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ and5′agccacacgcagctcattgtagaagg 3′ to amplify β-actin. First strand cDNA (5μl) was amplified in a total volume of 50 μl containing 0.4 μM primers,0.2 μM each dNTPs, 1× PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mMMgCl₂, 50 mM KCl, pH 8.3) and 1× Klentaq DNA polymerase (Clontech). Fiveμl of the PCR reaction was removed at 18, 20, and 22 cycles and used foragarose gel electrophoresis. PCR was performed using an MJ Researchthermal cycler under the following conditions: initial denaturation wasat 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. wascarried out for 2 min. After agarose gel electrophoresis, the bandintensities of the 283 bp β-actin bands from multiple tissues werecompared by visual inspection. Dilution factors for the first strandcDNAs were calculated to result in equal β-actin band intensities in alltissues after 22 cycles of PCR. Three rounds of normalization wererequired to achieve equal band intensities in all tissues after 22cycles of PCR.

[0176] To determine expression levels of the 8P1D4 gene, 5 μl ofnormalized first strand cDNA was analyzed by PCR using 25, 30, and 35cycles of amplification using the following primer pairs, which weredesigned with the assistance of (MIT; for details, see,www.genome.wi.mit.edu):

[0177] 5′ ACT TTG TTG ATG ACC AGG ATT GGA 3′ (SEQ ID NO. 28)

[0178] 5′ CAG AAC TTC AGC ACA CAC AGG AAC 3′ (SEQ ID NO. 29)

[0179] Semi quantitative expression analysis was achieved by comparingthe PCR products at cycle numbers that give light band intensities.

[0180] Results:

[0181] Several SSH experiments were conduced as described in theMaterials and Methods, supra, and led to the isolation of numerouscandidate gene fragment clones. All candidate clones were sequenced andsubjected to homology analysis against all sequences in the major publicgene and EST databases in order to provide information on the identityof the corresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or Northern analysis.

[0182] One of the cDNA clones, designated 8P1D4, was 436 bp in lengthand showed homology to an EST sequence in the NCI-CGAP tumor genedatabase. The full length cDNA encoding the 8P1D4 gene was subsequentlyisolated using this cDNA and re-named STEAP-1 (Example 2, below). The8P1D4 cDNA nucleotide sequence corresponds to nucleotide residues 150through 585 in the STEAP-1 cDNA sequence as shown in FIG. 1A. Anotherclone, designated 28P3E1, 561 bp in length showed homology to a numberof EST sequences in the NCI-CGAP tumor gene database or in otherdatabases. Part of the 28P3E1 sequence (356 bp) is identical to an ESTderived from human fetal tissue. After the full length STEAP-1 cDNA wasobtained and sequenced, it became apparent that this clone alsocorresponds to STEAP-1 (more specifically, to residues 622 through the3′ end of the STEAP-1 nucleotide sequence as shown in FIG. 1A).

[0183] Differential expression analysis by RT-PCR using primers derivedfrom the 8P1D4 cDNA clone showed that the 8P1D4 (STEAP-1) gene isexpressed at approximately equal levels in normal prostate and theLAPC-4 and LAPC-9 xenografts (FIG. 2, panel A). Further RT-PCRexpression analysis of first strand cDNAs from 16 normal tissues showedgreatest levels of 8P1D4 expression in prostate. Substantially lowerlevel expression in several other normal tissues (i.e., colon, ovary,small intestine, spleen and testis) was detectable only at 30 cycles ofamplification (FIG. 2, panels B and C).

Example 2 Isolation of Full Length STEAP-1 Encoding cDNA

[0184] The 436 bp 8P1D4 gene fragment (Example 1) was used to isolateadditional cDNAs encoding the 8P1D4/STEAP-1 gene. Briefly, a normalhuman prostate cDNA library (Clontech) was screened with a labeled probegenerated from the 436 bp 8P1D4 cDNA. One of the positive clones, clone10, is 1195 bp in length and encodes a 339 amino acid protein havingnucleotide and encoded amino acid sequences bearing no significanthomology to any known human genes or proteins (homology to a rat KidneyInjury Protein recently described in International ApplicationWO98/53071). The encoded protein contains at least 6 predictedtransmembrane motifs implying a cell surface orientation (see FIG. 1A,predicted transmembrane motifs underlined). These structural featuresled to the designation “STEAP”, for “Six Transmembrane EpithelialAntigen of the Prostate”. Subsequent identification of additional STEAPproteins led to the re-designation of the 8P1D4 gene product as“STEAP-1”. The STEAP-1 cDNA and encoded amino acid sequences are shownin FIG. 1A and correspond to SEQ ID NOS: 1 and 2, respectively. STEAP-1cDNA clone 10 has been deposited with the American Type CultureCollection (“ATCC”) (Mannassas, Va.) as plasmid 8P1D4 clone 10.1 on Aug.26, 1998 as ATCC Accession Number 98849. The STEAP-1 cDNA clone can beexcised therefrom using EcoRI/XbaI double digest (EcoRI at the 5′end,XbaI at the 3′end).

Example 3 STEAP-1 Gene and Protein Expression Analysis

[0185] In order to begin to characterize the biological characteristicsof STEAP-1, an extensive evaluation of STEAP-1 mRNA and STEAP-1 proteinexpression across a variety of human tissue specimens was undertaken.This evaluation included Northern blot, Western blot andimmunohistochemical analysis of STEAP-1 expression in a large number ofnormal human tissues, human prostate cancer xenografts and cell lines,and various other human cancer cell lines.

Example 3A Northern Blot Analysis of STEAP-1 mRNA Expression in NormalHuman Tissues

[0186] Initial analysis of STEAP-1 mRNA expression in normal humantissues was conducted by Northern blotting two multiple tissue blotsobtained from Clontech (Palo Alto, Calif.), comprising a total of 16different normal human tissues, using labeled STEAP-1 clone 10 as aprobe. RNA samples were quantitatively normalized with a β-actin probe.The results are shown in FIG. 3A. The highest expression level wasdetected in normal prostate, with an approximately 5-10 fold lower levelof expression detected in colon and liver. These northern blots showedtwo transcripts of approximately 1.4 kb and 4.0 kb, the former of whichcorresponds to the full length STEAP-1 clone 10 cDNA, which encodes theentire STEAP-1 open reading frame. The larger transcript was separatelycloned as a 3627 bp cDNA from a normal prostate library, the sequence ofwhich contains a 2399 bp intron (FIG. 4).

[0187] This initial analysis was extended by using the STEAP-1 clone 10probe to analyze an RNA dot blot matrix of 37 normal human tissues(Clontech, Palo Alto, Calif.; Human Master Blot™). The results are shownin FIG. 3B and show strong STEAP-1 expression only in prostate. Very lowlevel STEAP-1 RNA expression was detected in liver, lung, trachea andfetal liver tissue, at perhaps a 5-fold lower level compared toprostate. No expression was detected in any of the remaining tissues.Based on these analyses, significant STEAP-1 expression appears to beprostate specific in normal tissues.

Example 3B Northern Blot Analysis of STEAP-1 mRNA Expression in ProstateCancer Xenografts and Cell Lines

[0188] To analyze STEAP-1 expression in human cancer tissues and celllines, RNAs derived from human prostate cancer xenografts and anextensive panel of prostate and non-prostate cancer cell lines wereanalyzed by Northern blot using STEAP-1 cDNA clone 10 as probe. All RNAsamples were quantitatively normalized by ethiduim bromide staining andsubsequent analysis with a labeled β-actin probe.

[0189] The results, presented in FIG. 5, show high level STEAP-1expression in all the LAPC xenografts and all of the prostate cancercell lines. Expression in the LAPC-9 xenografts was higher compared tothe LAPC-4 xenografts, with no significant difference observed betweenandrogen-dependent and androgen-independent sublines (FIG. 5A).Expression in the LAPC-4 xenografts was comparable to expression innormal prostate. Lower levels of expression were detected in PrEC cells(Clonetics), which represent the basal cell compartment of the prostate.Analysis of prostate cancer cell lines showed highest expression levelsin LNCaP, an androgen dependent prostate carcinoma cell line.Significant expression was also detected in the androgen-independentcell lines PC-3 and DU145. High levels of STEAP expression were alsodetected in LAPC-4 and LAPC-9 tumors that were grown within the tibia ofmice as a model of prostate cancer bone metastasis (FIG. 5B).

[0190] Significantly, very strong STEAP-1 expression was also detectedin many of the non-prostate human cancer cell lines analyzed (FIG. 5A).Particularly high level expression was observed in RD-ES cells, an Ewingsarcoma (EWS) derived cell line. Additionally, very high levelexpression was also detected in several of the colon cancer cell lines(e.g., CaCo-2, LoVo, T84 and Colo-205), bladder carcinoma cell lines(e.g., SCABER, UM-UC-3, TCCSUP and 5637), ovarian cancer cell lines(e.g., OV-1063 and SW 626) and pancreatic cancer cell lines (e.g., HPAC,Capan-1, PANC-1 and BxPC-3). These results, combined with the absence ofstrong expression in the corresponding normal tissues (FIG. 3), indicatethat STEAP-1 may be generally up-regulated in these types (as well asother types) of human cancers.

Example 3C Western Blot Analysis of STEAP-1 Protein Expression inProstate and Other Cancers

[0191] A 15 mer peptide corresponding to amino acid residues 14 through28 of the STEAP-1 amino acid sequence as shown in FIG. 1A(WKMKPRRNLEEDDYL)(SEQ ID NO: 2) was synthesized and used to immunizesheep for the generation of sheep polyclonal antibodies towards theamino-terminus of the protein (anti-STEAP-1) as follows. The peptide wasconjugated to KLH (keyhole limpet hemocyanin). The sheep was initiallyimmunized with 400 μg of peptide in complete Freund's adjuvant. Theanimal was subsequently boosted every two weeks with 200 μg of peptidein incomplete Freund's adjuvant. Anti-STEAP antibody wasaffinity-purified from sheep serum using STEAP peptide coupled toaffi-gel 10 (Bio Rad). Purified antibody is stored in phosphate-bufferedsaline with 0.1% sodium azide.

[0192] To test antibody specificity, the cDNA of STEAP-1 was cloned intoa retroviral expression vector (pSRαtkneo, Muller et al., 1991, MCB11:1785). NIH 3T3 cells were infected with retroviruses encoding STEAP-1and were selected in G418 for 2 weeks. Western blot analysis of proteinextracts of infected and un-infected NIH 3T3 cells showed expression ofa protein with an apparent molecular weight of 36 kD only in theinfected cells (FIG. 6, lanes marked “3T3 STEAP” AND “3T3”).

[0193] The anti-STEAP-1 polyclonal antibody was used to probe Westernblots of cell lysates prepared from a variety of prostate cancerxenograft tissues, prostate cancer cell lines and other non-prostatecancer cell lines. Protein samples (20 μg each) were quantitativelynormalized by probing the blots with an anti-Grb-2 antibody.

[0194] The results are shown in FIG. 6. STEAP-1 protein was detected inall of the LAPC prostate cancer xenografts, all of the prostate cancercell lines, a primary prostate cancer specimen and its matched normalprostate control. Highest STEAP-1 protein expression was detected in theLAPC-9 xenograft and in LNCaP cells, in agreement with the Northern blotanalysis described immediately above. High level expression was alsoobserved in the bladder carcinoma cell line UM-UC-3. Expression in othercancer cell lines was also detectable (FIG. 6).

Example 3D Immunohistochemical Analysis of STEAP-1 Protein Expression inProstate Tumor Biopsy and Surgical Specimens

[0195] To determine the extent of STEAP-1 protein expression in clinicalmaterials, tissue sections were prepared from a variety of prostatecancer biopsies and surgical samples for immunohistochemical analysis.Tissues were fixed in 10% formalin, embedded in paraffin, and sectionedaccording to standard protocol. Formalin-fixed, paraffin-embeddedsections of LNCaP cells were used as a positive control. Sections werestained with an anti-STEAP-1 polyclonal antibody directed against aSTEAP-1 N-terminal epitope (as described immediately above). LNCaPsections were stained in the presence of an excess amount of the STEAP-1N-terminal peptide immunogen used to generate the polyclonal antibody(peptide 1) or a non-specific peptide derived from a distinct region ofthe STEAP-1 protein (peptide 2; YQQVQQNKEDAWIEH).

[0196] The results are shown in FIG. 8. LNCaP cells showed uniformlystrong peri-cellular staining in all cells (FIG. 8b). Excess STEAPN-terminal peptide (peptide 1) was able to competitively inhibitantibody staining (FIG. 8a), while peptide 2 had no effect (FIG. 8b).Similarly, uniformly strong peri-cellular staining was seen in theLAPC-9 (FIG. 8f) and LAPC-4 prostate cancer xenografts (data not shown).These results are clear and suggest that the staining is STEAP specific.Moreover, these results visually localize STEAP to the plasma membrane,corroborating the biochemical findings presented in Example 4 below.

[0197] The results obtained with the various clinical specimens are showin FIG. 8c (normal prostate tissue), FIG. 8d (grade 3 prostaticcarcinoma), and FIG. 8e (grade 4 prostatic carcinoma), and are alsoincluded in the summarized results shown in TABLE 1. Light to strongstaining was observed in the glandular epithelia of all prostate cancersamples tested as well as in all samples derived from normal prostate orbenign disease. The signal appears to be strongest at the cell membraneof the epithelial cells, especially at the cell-cell junctions (FIGS.8c, d and e) and is also inhibited with excess STEAP N-terminal peptide1 (data not shown). Some basal cell staining is also seen in normalprostate (FIG. 8c), which is more apparent when examining atrophicglands (data not shown). STEAP-1 seems to be expressed at all stages ofprostate cancer since lower grades (FIG. 8d), higher grades (FIG. 8e)and metastatic prostate cancer (represented by LAPC-9; FIG. 8f) allexhibit strong staining.

[0198] Immunohistochemical staining of a large panel of normalnon-prostate tissues showed no detectable STEAP-1 expression in 24 of 27of these normal tissues (Table 1). Only three tissue samples showed somedegree of anti-STEAP-1 staining. In particular, normal bladder exhibitedlow levels of cell surface staining in the transitional epithelium (FIG.8g). Pancreas and pituitary showed low levels of cytoplasmic staining(Table 1). It is unclear whether the observed cytoplasmic staining isspecific or is due to non-specific binding of the antibody, sincenorthern blotting showed little to no STEAP-1 expression in pancreas(FIG. 3). Normal colon, which exhibited higher mRNA levels than pancreasby Northern blotting (FIG. 3), exhibited no detectable staining withanti-STEAP antibodies (FIG. 8h). These results indicate that cellsurface expression of STEAP-1 in normal tissues appears to be restrictedto prostate and bladder. TABLE 1 IMMUNOHISTOCHEMICAL STAINING OF HUMANTISSUES WITH ANTI-STEAP-1 POLYCLONAL ANTIBODY STAINING INTENSITY TISSUENONE cerebellum, cerebral cortex, spinal cord, heart, skeletal muscle,artery, thymus, spleen, bone marrow, lymph node, lung, colon, liver,stomach, kidney, testis, ovary, fallopian tubes, placenta, uterus,breast, adrenal gland, thyroid gland, skin, bladder (3/5) LIGHT TObladder (2/5), pituitary gland (cytoplasmic), pancreas MODERATE(cytoplasmic), BPH (3/5), prostate cancer (3/10) STRONG prostate (2/2),BPH (2/5), prostate cancer** (7/10)

Example 4 Biochemical Characterization of STEAP-1 Protein

[0199] To initially characterize the STEAP-1 protein, cDNA clone 10 (SEQID NO. 1) was cloned into the pcDNA 3.1 Myc-His plasmid (Invitrogen),which encodes a 6His tag at the carboxyl-terminus, transfected into 293Tcells, and analyzed by flow cytometry using anti-His monoclonal antibody(His-probe, Santa Cruz) as well as the anti-STEAP-1 polyclonal antibodydescribed above. Staining of cells was performed on intact cells as wellas permeabilized cells. The results indicated that only permeabilizedcells stained with both antibodies, suggesting that both termini of theSTEAP-1 protein are localized intracellularly. It is therefore possiblethat one or more of the STEAP-1 protein termini are associated withintracellular organelles rather than the plasma membrane.

[0200] To determine whether STEAP-1 protein is expressed at the cellsurface, intact STEAP-1-transfected 293T cells were labeled with abiotinylation reagent that does not enter live cells. STEAP-1 was thenimmunoprecipitated from cell extracts using the anti-His and anti-STEAPantibodies. SV40 large T antigen, an intracellular protein that isexpressed at high levels in 293T cells, and the endogenous cell surfacetransferrin receptor were immunoprecipitated as negative and positivecontrols, respectively. After immunoprecipitation, the proteins weretransferred to a membrane and visualized with horseradishperoxidase-conjugated streptavidin. The results of this analysis areshown in FIG. 7. Only the transferrin receptor (positive control) andSTEAP-1 were labeled with biotin, while the SV40 large T antigen(negative control) was not detectably labeled (FIG. 7A). Since only cellsurface proteins are labeled with this technique, it is clear from theseresults that STEAP-1 is a cell surface protein. Combined with theresults obtained from the flow cytometric analysis, it is clear thatSTEAP-1 is a cell surface protein with intracellular amino- andcarboxyl-termini.

[0201] Furthermore, the above results together with the STEAP-1secondary structural predictions, shows that STEAP-1 is a type IIIamembrane protein with a molecular topology of six potentialtransmembrane domains, 3 extracellular loops, 2 intracellular loops andtwo intracellular termini. A schematic representation of STEAP-1 proteintopology relative to the cell membrane is shown in FIG. 1B.

[0202] In addition, prostate, bladder and colon cancer cells weredirectly analyzed for cell surface expression of STEAP-1 bybiotinylation studies. Briefly, biotinylated cell surface proteins wereaffinity purified with streptavidin-gel and probed with the anti-STEAP-1polyclonal antibody described above. Western blotting of thestreptavidin purified proteins clearly show cell surface biotinylationof endogenous STEAP-1 in all prostate (LNCaP, PC-3, DU145), bladder(UM-UC-3, TCCSUP) and colon cancer (LoVo, CoIo) cells tested, as well asin NIH 3T3 cells infected with a STEAP-1 encoding retrovirus, but not innon-expressing NIH 3T3 cells used as a negative control (FIG. 7B). In afurther negative control, STEAP-1 protein was not detected instreptavidin precipitates from non-biotinylated STEAP expressing cells(FIG. 7B).

Example 5 Identification and Structural Analysis of STEAP-2 and OtherHuman STEAP Family Members

[0203] STEAP-1 has no homology to any known human genes. In an attemptto identify additional genes that are homologous to STEAP-1, the proteinsequence of STEAP-1 was used as an electronic probe to identify familymembers in the public EST (expression sequence tag) database (dbest).Using the “tblastn” function in NCBI (National Center for BiotechnologyInformation), the dbest database was queried with the STEAP-1 proteinsequence. This analysis revealed additional putative STEAP-1 homologuesor STEAP family members, as further described below.

[0204] In addition, applicants cloning experiments also identified aSTEAP-1 related SSH cDNA fragment, clone 98P4B6. This clone was isolatedfrom SSH cloning using normal prostate cDNA as tester and LAPC-4 AD cDNAas driver. A larger partial sequence of the 98P4B6 clone wassubsequently isolated from a normal prostate library; this clone encodesan ORF of 173 amino acids with close homology to the primary structureof STEAP-1, and thus was designated STEAP-2.

[0205] The STEAP-2 partial nucleotide and encoded ORF amino acidsequences are shown in FIG. 9. An amino acid alignment of the STEAP-1and partial STEAP-2 primary structures is shown in FIG. 11A. STEAP-1 and-2 share 61% identity over their 171 amino acid residue overlap (FIG.11A). Despite their homology, STEAP-1 and -2 show significantlydivergent expression patterns in normal and cancerous tissues and cells,and also map to distinct locations on opposite arms of human chromosome7 (see Examples 6 and 7 below).

[0206] Two ESTs identified by electronic probing with the STEAP-1protein sequence, All 39607 and R80991, encode ORFs bearing closehomology to the STEAP-1 and STEAP-2 sequences and thus appear torepresent two additional STEAPs. Their nucleotide sequences arereproduced in FIG. 10 and their encoded ORF STEAP-like amino acidsequences are shown in FIG. 11B. The ORFs encoded by these ESTs areunique but show very clear structural relationships to both STEAP-1 andSTEAP-2, particularly in the conserved transmembrane domains.Accordingly these ESTs appear to correspond to distinct STEAP familymembers and have thus been designated as STEAP-3 (corresponding to All39607) and STEAP-4 (corresponding to R80991).

[0207] An amino acid alignment of the complete STEAP-1 protein sequencewith the predicted partial STEAP-2, STEAP-3 and STEAP-4 amino acidsequences is shown in FIG. 11B. This alignment shows a close structuralsimilarity between all four STEAP family proteins, particularly in thepredicted transmembrane domains, even though only partial sequenceinformation was available for three of them. The STEAP-3 and STEAP-4proteins appear to be more closely related to STEAP-2 than to STEAP-1 oreach other. Specifically, STEAP-3 shows 50% identity and 69% homology toSTEAP-2, versus 37% identity and 63% homology to STEAP-1. STEAP-4 shows56% identity and 87% homology to STEAP-2, versus 42% identity and 65%homology to STEAP-1. STEAP-3 and STEAP-4 are 38% identical and 57%homologous to each other. These figures are estimates based uponincomplete sequence information. However, these figures suggestconservation of at least some of the transmembrane domains, suggestingcommon topological characteristics if not functional characteristics.

Example 6 Expression Analysis of STEAP-2 and Other Human STEAP FamilyMembers Example 6A Tissue Specific Expression of STEAP Family Members inNormal Human Tissues

[0208] Expression analysis of STEAP family members in normal tissues wasperformed by RT-PCR. All STEAP family members appeared to exhibit tissuerestricted expression patterns. AI139607 expression is detected inplacenta and prostate after 25 cycles of amplification (FIG. 12). After30 cycles, AI139607 expression is also detected in other tissues. R80991expression is highest in normal liver, although expression is alsodetected in other tissues after 30 cycles of amplification (FIG. 13).Neither R80991, nor AI139607 expression was detected in the LAPCprostate cancer xenografts by RT-PCR.

[0209] RT-PCR analysis of STEAP-2 shows expression in all the LAPCprostate cancer xenografts and in normal prostate (FIG. 14, panel A).Analysis of 8 normal human tissues shows prostate-specific expressionafter 25 cycles of amplification (FIG. 14, panel B). Lower levelexpression in other tissues was detected only after 30 cycles ofamplification. Northern blotting for STEAP-2 shows a pattern of 2transcripts (approximately 3 and 8 kb in size) expressed only inprostate (and at significantly lower levels in the LAPC xenografts),with no detectable expression in any of the 15 other normal humantissues analyzed (FIG. 15, panel C). Thus, STEAP-2 expression in normalhuman tissues appears to be highly prostate-specific.

Example 6B Expression of STEAP-2 in Various Cancer Cell Lines

[0210] The RT-PCR results above suggested that the different STEAPfamily members exhibit different tissue expression patterns.Interestingly, STEAP-2, which appears very prostate-specific, seems tobe expressed at lower levels in the LAPC xenografts. This is in contrastto STEAP-1, which is highly expressed in both normal and malignantprostate tissue.

[0211] To better characterize this suggested difference in the STEAP-2prostate cancer expression profile (relative to STEAP-1), Northernblotting was performed on RNA derived from the LAPC xenografts, as wellas several prostate and other cancer cell lines, using a STEAP-2specific probe (labeled cDNA clone 98P4B6). The results are shown inFIG. 16 and can be summarized as follows. STEAP-2 is highly expressed innormal prostate and in some of the prostate cancer xenografts and celllines. More particularly, very strong expression was observed in theLAPC-9 AD xenograft and the LNCaP cells. Significantly attenuated or noexpression was observed in the other prostate cancer xenografts and celllines. Very strong expression was also evident in the Ewing Sarcoma cellline RD-ES. Unlike STEAP-1, which is highly expressed in cancer celllines derived from bladder, colon, pancreatic and ovarian tumors,STEAP-2 showed low to non-detectable expression in these same cell lines(compare with FIG. 5). Interestingly, STEAP-2 was also non-detectable inPrEC cells, which are representative of the normal basal cellcompartment of the prostate. These results suggests that expression ofSTEAP-1 and STEAP-2 are differentially regulated. While STEAP-1 may be agene that is generally up-regulated in cancer, STEAP-2 may be a genethat is more restricted to normal prostate and prostate cancer.

Example 7 Chromosomal Localization of STEAP Genes

[0212] The chromosomal localization of STEAP-1 was determined using theGeneBridge 4 Human/Hamster radiation hybrid (RH) panel (Walter et al.,1994, Nat. Genetics 7:22) (Research Genetics, Huntsville Ala.), whileSTEAP-2 and the STEAP homologues were mapped using the Stanford G3radiation hybrid panel (Stewart et al., 1997, Genome Res. 7:422).

[0213] The following PCR primers were used for STEAP-1:

[0214] 8P1D4.1 5′ ACTTTGTTGATGACCAGGATTGGA 3′ (SEQ ID NO: 4)

[0215] 8P1D4.2 5′ CAGAACTTCAGCACACACAGGAAC 3′ (SEQ ID NO: 5)

[0216] The resulting STEAP-1 mapping vector for the 93 radiation hybridpanel DNAs(210000020110101000100000010111010122100011100111011010100010001000101001021000001111001010000), and the mapping program available at theInternet address<http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl>, localized theSTEAP-1 gene to chromosome 7p22.3, telomeric to D7S531.

[0217] The following PCR primers were used for 98P4B6/STEAP-2:

[0218] 98P4B6.1 5′ GACTGAGCTGGAACTGGAATTTGT 3′ (SEQ ID NO: 17)

[0219] 98P4B6.2 5′ TTTGAGGAGACTTCATCTCACTGG 3′ (SEQ ID NO: 18)

[0220] The resulting vector(00000100100000000000000000000000100100000000001000100000000000001000010101010010011), and the mapping program available at theinternet address<http://www-shgc.stanford.edu/RH/rhserverformew.html>maps the 98P4B6 (STEAP-2) gene to chromosome 7q21.

[0221] The following PCR primers were used for AI139607:

[0222] AI139607.1 5′ TTAGGACAACTTGATCACCAGCA 3′ (SEQ ID NO: 13)

[0223] AI139607.2 5′TGTCCAGTCCAAACTGGGTTATTT3′ (SEQ ID NO: 14)

[0224] The resulting vector(00000000100000000000000000001000100000200000001000100000001000000100010001010000010), and the mapping program available at theinternet address <http://www-shgc.stanford.edu/RH/rhserverformew.html>,maps AI139607 to chromosome 7q21.

[0225] The following PCR primers were used for R80991:

[0226] R80991.3 5′ ACAAGAGCCACCTCTGGGTGAA 3′ (SEQ ID NO: 15)

[0227] R80991.4 5′ AGTTGAGCGAGTTTGCAATGGAC 3′ (SEQ ID NO: 16)

[0228] The resulting vector(00000000000200001020000000010000000000000000000010000000001000011100000001001000001), and the mapping program available atthe internet address<http://www-shgc.stanford.edu/RH/rhserverformew.html> maps R80991 tochromosome 2q14-q21, near D2S2591.

[0229] In summary, the above results show that three of the putativehuman STEAP family members localize to chromosome 7, as is schematicallydepicted in FIG. 17. In particular, the STEAP-1 gene localizes to thefar telomeric region of the short arm of chromosome 7, at 7p22.3, whileSTEAP-2 and AI139607 localize to the long arm of chromosome 7, at 7q21(FIG. 17). R80991 maps to chromosome 2q14-q21.

Example 8 Identification of Intron-Exon Boundaries of STEAP-1

[0230] Genomic clones for STEAP-1 were identified by searching GenBankfor BAC clones containing STEAP-1 sequences, resulting in theidentification of accession numbers AC004969 (PAC DJ1121E10) andAC005053 (BAC RG04D11). Using the sequences derived from the PAC and BACclones for STEAP the intron-exon boundaries were defined (FIG. 18). Atotal of 4 exons and 3 introns were identified within the coding regionof the STEAP gene. Knowledge of the exact exon-intron structure of theSTEAP-1 gene may be used for designing primers within intronic sequenceswhich in turn may be used for genomic amplification of exons. Suchamplification permits single-stranded conformational polymorphism (SSCP)analysis to search for polymorphisms associated with cancer. Mutant orpolymorphic exons may be sequenced and compared to wild type STEAP. Suchanalysis may be useful to identify patients who are more susceptible toaggressive prostate cancer, as well as other types of cancer,particularly colon, bladder, pancreatic, ovarian, cervical andtesticular cancers.

[0231] Southern blot analysis shows that the STEAP-1 gene exists inseveral species including mouse (FIG. 19). Therefore, a mouse BAClibrary (Mouse ES 129-V release 1, Genome Systems, FRAC-4431) wasscreened with the human cDNA for STEAP-1 (clone 10, Example 2). Onepositive clone, 12P11, was identified and confirmed by southern blotting(FIG. 20). The intron-exon boundary information for human STEAP may beused to identify the mouse STEAP-1 coding sequences.

[0232] The mouse STEAP-1 genomic clone may be used to study thebiological role of STEAP-1 during development and tumorigenesis.Specifically, the mouse genomic STEAP-1 clone may be inserted into agene knock-out (K/O) vector for targeted disruption of the gene in mice,using methods generally known in the art. In addition, the role of STEAPin metabolic processes and epithelial cell function may be elucidated.Such K/O mice may be crossed with other prostate cancer mouse models,such as the TRAMP model (Greenberg et al., 1995, PNAS 92:3439), todetermine whether STEAP influences the development and progression ofmore or less aggressive and metastatic prostate cancers.

[0233] This application claims the benefit of the filing dates of U.S.Provisional Patent Applications No. 06/087,520 filed Jun. 1, 1998 andNo. 06/091,183 filed Jun. 30, 1998 under the provisions of 37 USC119(e), the contents of which are incorporated by reference herein intheir entireties.

[0234] Throughout this application, various publications are referencedwithin parentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

[0235] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1 32 1 1195 DNA Homo Sapiens 1 ccgagactca cggtcaagct aaggcgaagagtgggtggct gaagccatac tattttatag 60 aattaatgga aagcagaaaa gacatcacaaaccaagaaga actttggaaa atgaagccta 120 ggagaaattt agaagaagac gattatttgcataaggacac gggagagacc agcatgctaa 180 aaagacctgt gcttttgcat ttgcaccaaacagcccatgc tgatgaattt gactgccctt 240 cagaacttca gcacacacag gaactctttccacagtggca cttgccaatt aaaatagctg 300 ctattatagc atctctgact tttctttacactcttctgag ggaagtaatt caccctttag 360 caacttccca tcaacaatat ttttataaaattccaatcct ggtcatcaac aaagtcttgc 420 caatggtttc catcactctc ttggcattggtttacctgcc aggtgtgata gcagcaattg 480 tccaacttca taatggaacc aagtataagaagtttccaca ttggttggat aagtggatgt 540 taacaagaaa gcagtttggg cttctcagtttcttttttgc tgtactgcat gcaatttata 600 gtctgtctta cccaatgagg cgatcctacagatacaagtt gctaaactgg gcatatcaac 660 aggtccaaca aaataaagaa gatgcctggattgagcatga tgtttggaga atggagattt 720 atgtgtctct gggaattgtg ggattggcaatactggctct gttggctgtg acatctattc 780 catctgtgag tgactctttg acatggagagaatttcacta tattcagagc aagctaggaa 840 ttgtttccct tctactgggc acaatacacgcattgatttt tgcctggaat aagtggatag 900 atataaaaca atttgtatgg tatacacctccaacttttat gatagctgtt ttccttccaa 960 ttgttgtcct gatatttaaa agcatactattcctgccatg cttgaggaag aagatactga 1020 agattagaca tggttgggaa gacgtcaccaaaattaacaa aactgagata tgttcccagt 1080 tgtagaatta ctgtttacac acatttttgttcaatattga tatattttat caccaacatt 1140 tcaagtttgt atttgttaat aaaatgattattcaaggaaa aaaaaaaaaa aaaaa 1195 2 339 PRT Artificial Sequence DNA 2 MetGlu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10 15Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70 7580 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His 85 9095 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu115 120 125 Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His AsnGly 130 135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp MetLeu Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe AlaVal Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg SerTyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln GlnAsn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met GluIle Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu Ala LeuLeu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp Ser Leu ThrTrp Arg Glu Phe His Tyr Ile Gln Ser Lys 245 250 255 Leu Gly Ile Val SerLeu Leu Leu Gly Thr Ile His Ala Leu Ile Phe 260 265 270 Ala Trp Asn LysTrp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro 275 280 285 Pro Thr PheMet Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile Phe 290 295 300 Lys SerIle Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile 305 310 315 320Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile Cys 325 330335 Ser Gln Leu 3 111 DNA Homo sapiens 3 ggcggaggcg gaggcggagggcgaggggcg gggagcgccg cctggagcgc ggcaggtcat 60 attgaacatt ccagatacctatcattactc gatgctgttg ataacagcaa g 111 4 24 DNA Artificial SequencePrimer 4 actttgttga tgaccaggat tgga 24 5 24 DNA Artificial SequencePrimer 5 cagaacttca gcacacacag gaac 24 6 3627 DNA Homo sapiens 6ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga 60gtgggtggct gaagccatac tattttatag aattaatgga aagcagaaaa gacatcacaa 120accaagaaga actttggaaa atgaagccta ggagaaattt agaagaagac gattatttgc 180ataaggacac gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa 240cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc 300cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact tttctttaca 360ctcttctgag ggaagtaatt caccccttag caacttccca tcaacaatat ttttataaaa 420ttccaatcct ggtcatcaac aaagtcttgc caatggtttc catcactctc ttggcattgg 480tttacctgcc aggtgtgata gcagcaattg tccaacttca taatggaacc aagtataaga 540agtttccaca ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcagtt 600tcttttttgc tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca 660gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa gatgcctgga 720ttgagcatga tgtttggaga atggagattt atgtgtctct gggaattgtg ggattggcaa 780tactggctct gttggctgtg acatctattc catctgtgag tgactctttg acatggagag 840aatttcacta tattcaggta aataatatat aaaataaccc taagaggtaa atcttctttt 900tgtgtttatg atatagaata tgttgacttt accccataaa aaataacaaa tgtttttcaa 960cagcaaagat cttatacttg ttccaattaa taatgtgctc tcctgttgtt ttccctattg 1020cttctaatta ggacaagtgt ttcctagaca taaataaaag gcattaaaat attctttgtt 1080tttttttttt tgtttgtttg ttttttgttt gtttgtttgt ttttttgaga tgaagtctcg 1140ctctgttgcc catgctggag tacagtggca cgatctcggc tcactgcaac ctgcgcctcc 1200tgggttcagg cgattctctt gcctcagcct cctgagtagc tgggattaca ggcacccatc 1260accatgtcca gctaattttt gtatttttag tagagacagg gttttcccat gttggccagg 1320ctggtctcga tctcctgacc tcaaatgatc cgcccacctc ggcctcccaa agtgctggga 1380tgacagttgt gagccaccac actcagcctg ctctttctaa tatttgaaac ttgttagaca 1440atttgctacc catctaatgt gatattttag gaatccaata tgcatggttt attatttctt 1500aaaaaaaata ttcttttacc tgtcacctga atttagtaat gccttttatg ttacacaact 1560tagcactttc cagaaacaaa aactctctcc ttgaaataat agagttttta tctaccaaag 1620atatgctagt gtctcatttc aaaggctgct ttttccagct tacattttat atacttactc 1680acttgaagtt tctaaatatt cttgtaattt taaaactatc tcagatttac tgaggtttat 1740cttctggtgg tagattatcc ataagaagag tgatgtgcca gaatcactct gggatccttg 1800tctgacaaga ttcaaaggac taaatttaat tcagtcatga acactgccaa ttaccgttta 1860tgggtagaca tctttggaaa tttccacaag gtcagacatt cgcaactatc ccttctacat 1920gtccacacgt atactccaac actttattag gcatctgatt agtttggaaa gtatgcctcc 1980atctgaatta gtccagtgtg gcttagagtt ggtacaacat tctcacagaa tttcctaatt 2040ttgtaggttc agcctgataa ccactggagt tctttggtcc tcattaaata gctttcttca 2100cacattgctc tgcctgttac acatatgatg aacactgctt tttagacttc attaggaatt 2160taggactgca tcttgacaac tgagcctatt ctactatatg tacaatacct agcccataat 2220aggtatacaa tacacatttg gtaaaactaa ttttcaacca atgacatgta tttttcaact 2280agtaacctag aaatgtttca cttaaaatct gagaactggt tacactacaa gttaccttgg 2340agattcatat atgaaaacgc aaacttagct atttgattgt attcactggg acttaagaat 2400gcgcctgaat aattgtgagt tcgatttgtt ctggcaggct aatgaccatt tccagtaaag 2460tgaatagagg tcagaagtcg tataaaagag gtgttgtcag aacaccgttg agattacata 2520ggtgaacaac tatttttaag caactttatt tgtgtagtga caaagcatcc caatgcaggc 2580tgaaatgttt catcacatct ctggatctct ctattttgtg cagacattga aaaaattgtt 2640catattattt ccatgttatc agaatatttg attttttaaa aacataggcc aagttcattc 2700acttcattat tcatttatca aaatcagagt gaatcacatt agtcgccttc acaactgata 2760aagatcactg aagtcaaatt gatttttgct ataatcttca atctacctat atttaattga 2820gaatctaaaa tgtacaaatc attgtgttga ttctgcagtg atcctgctat aagtaagact 2880cagtccctga ttttaggtat cctgtgaaaa gcagaattaa gacaaataca caagagacaa 2940agcacaaaaa ataaatatca taaggggatg aacaaaatgg tggagaaaga gtagacaaag 3000tttttgatca cctgccttca aagaaaggct gtgaattttg ttcacttaga cagcttggag 3060acaagaaatt acccaaaagt aaggtgagga ggataggcaa aaagagcaga aagatgtgaa 3120tggacattgt tgagaaatgt gataggaaaa caatcataga taaaggattt ccaagcaaca 3180gagcatatcc agatgaggta ggatgggata aactcttatt gaaccaatct tcaccaattt 3240tgtttttctt ttgcagagca agctaggaat tgtttccctt ctactgggca caatacacgc 3300attgattttt gcctggaata agtggataga tataaaacaa tttgtatggt atacacctcc 3360aacttttatg atagctgttt tccttccaat tgttgtcctg atatttaaaa gcatactatt 3420cctgccatgc ttgaggaaga agatactgaa gattagacat ggttgggaag acgtcaccaa 3480aattaacaaa actgagatat gttcccagtt gtagaattac tgtttacaca catttttgtt 3540caatattgat atattttatc accaacattt caagtttgta tttgttaata aaatgattat 3600tcaaggaaaa aaaaaaaaaa aaaaaaa 3627 7 519 DNA Homo sapiens 7 gacttttacaaaattcctat agagattgtg aataaaacct tacctatagt tgccattact 60 ttgctctccctagtatacct cgcaggtctt ctggcagctg cttatcaact ttattacggc 120 accaagtataggagatttcc accttggttg gaaacctggt tacagtgtag aaaacagctt 180 ggattactaagttttttctt cgctatggtc catgttgcct acagcctctg cttaccgatg 240 agaaggtcagagagatattt gtttctcaac atggcttatc agcaggttca tgcaaatatt 300 gaaaactcttggaatgagga agaagtttgg agaattgaaa tgtatatctc ctttggcata 360 atgagccttggcttactttc cctcctggca gtcacttcta tcccttcagt gagcaatgct 420 ttaaactggagagaattcag ttttattcag tctacacttg gatatgtcgc tctgctcata 480 agtactttccatgttttaat ttatggatgg aaacgagct 519 8 173 PRT Homo sapiens 8 Asp Phe TyrLys Ile Pro Ile Glu Ile Val Asn Lys Thr Leu Pro Ile 1 5 10 15 Val AlaIle Thr Leu Leu Ser Leu Val Tyr Leu Ala Gly Leu Leu Ala 20 25 30 Ala AlaTyr Gln Leu Tyr Tyr Gly Thr Lys Tyr Arg Arg Phe Pro Pro 35 40 45 Trp LeuGlu Thr Trp Leu Gln Cys Arg Lys Gln Leu Gly Leu Leu Ser 50 55 60 Phe PhePhe Ala Met Val His Val Ala Tyr Ser Leu Cys Leu Pro Met 65 70 75 80 ArgArg Ser Glu Arg Tyr Leu Phe Leu Asn Met Ala Tyr Gln Gln Val 85 90 95 HisAla Asn Ile Glu Asn Ser Trp Asn Glu Glu Glu Val Trp Arg Ile 100 105 110Glu Met Tyr Ile Ser Phe Gly Ile Met Ser Leu Gly Leu Leu Ser Leu 115 120125 Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asn Ala Leu Asn Trp Arg 130135 140 Glu Phe Ser Phe Ile Gln Ser Thr Leu Gly Tyr Val Ala Leu Leu Ile145 150 155 160 Ser Thr Phe His Val Leu Ile Tyr Gly Trp Lys Arg Ala 165170 9 322 DNA Homo sapiens 9 ggtcgacttt tcctttattc ctttgtcaga gatctgattcatccatatgc tagaaaccaa 60 cagagtgact tttacaaaat tcctatagag attgtgaataaaaccttacc tatagttgcc 120 attactttgc tctccctagt ataccttgca ggtcttctggcagctgctta tcaactttat 180 tacggcacca agtataggag atttccacct tggttggaaacctggttaca gtgtagaaaa 240 cagcttggat tactaagttg tttcttcgct atggtccatgttgcctacag cctctgctta 300 ccgatgagaa ggtcagagag at 322 10 183 DNA Homosapiens 10 tttgcagctt tgcagatacc cagactgagc tggaactgga atttgtcttcctattgactc 60 tacttcttta aaagcggctg cccattacat tcctcagctg tccttgcagttaggtgtaca 120 tgtgactgag tgttggccag tgagatgaag tctcctcaaa ggaaggcagcatgtgtcctt 180 ttt 183 11 448 DNA Homo sapiens 11 aagaaggaga atccatttagcacctcctca gcctggctca gtgattcata tgtggctttg 60 ggaatacttg ggttttttctgtttgtactc ttgggaatca cttctttgcc atctgttagc 120 aatgcagtca actggagagagttccgattt gtccagtcca aactgggtta tttgaccctg 180 atcttgtgta cagcccacaccctggtgtac ggtgggaaga gattcctcag cccttcaaat 240 ctcagatggt atcttcctgcagcctacgtg ttagggctta tcattccttg cactgtgctg 300 gtgatcaagt ttgtcctaatcatgccatgt gtagacaaca cccttacaag gatccgccag 360 ggctgggaaa ggaactcaaaacactagaaa aagcattgaa tggaaaatca atatttaaaa 420 caaagttcaa tttagctggaaaaaaaaa 448 12 401 DNA Homo sapiens misc_feature (1)...(401) n = A,T,Cor G 12 ggccgcggca nccgctacga cctggtcaac ctggcagtca agcaggtcttggccanacaa 60 gagccacctc tgggtgaagg aggaggtctg gcggatggag atctacctctccctgggagt 120 gctggccctc ggcacgttgt ccctgctggc cgtgacctca ctgccgtccattgcaaactc 180 gctcaactgg agggagttca gcttcgttca gtcctcactg ggctttgtggccntcgtgct 240 gagcacactn cacacgctca cctacggctg gacccgcgcc ttcgaggagagccgctacaa 300 gttctacctn cctcccacct tcacgntcac gctgctggtg ccctgcgttcgttcatcctg 360 ggccaaagcc ctgtttntac tgccttgcat tcagccgnag a 401 13 23DNA Artificial Sequence RT-PCR Primer AI139607.1 13 ttaggacaacttgatcacca gca 23 14 24 DNA Artificial Sequence RT-PCR primer AI139607.214 tgtccagtcc aaactgggtt attt 24 15 23 DNA Artificial Sequence RT-PCRprimer R80991.1 15 agggagttca gcttcgttca gtc 23 16 24 DNA ArtificialSequence RT-PCR primer R80991.2 16 ggtagaactt gtagcggctc tcct 24 17 24DNA Artificial Sequence RT-PCR primer 98P4B6.1 17 gactgagctg gaactggaatttgt 24 18 24 DNA Artificial Sequence RT-PCR primer 98P4B6.2 18tttgaggaga cttcatctca ctgg 24 19 22 PRT Artificial Sequence STEAP-1peptide 19 Arg Glu Val Ile His Pro Leu Ala Thr Ser His Gln Gln Tyr PheTyr 1 5 10 15 Lys Ile Pro Ile Leu Val 20 20 34 PRT Artificial SequenceSTEAP-1 peptide 20 Arg Arg Ser Tyr Arg Tyr Lys Leu Leu Asn Trp Ala TyrGln Gln Val 1 5 10 15 Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His AspVal Trp Arg Met 20 25 30 Glu Ile 21 15 PRT Artificial Sequence STEAP-1PEPTIDE 21 Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro Pro Thr Phe 15 10 15 22 14 DNA Artificial Sequence cDNA Synthesis primer 22ttttgtacaa gctt 14 23 44 DNA Artificial Sequence DNA Adaptor 1 23ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44 24 42 DNA ArtificialSequence DNA Adaptor 2 24 gtaatacgac tcactatagg gcagcgtggt cgcggccgag gt42 25 22 DNA Artificial Sequence PCR primer 1 25 ctaatacgac tcactatagggc 22 26 22 DNA Artificial Sequence Nested primer (NP) 1 26 tcgagcggccgcccgggcag gt 22 27 20 DNA Artificial Sequence Nested primer (NP) 2 27agcgtggtcg cggccgaggt 20 28 24 DNA Artificial Sequence RT-PCR primer 1A28 actttgttga tgaccaggat tgga 24 29 24 DNA Artificial Sequence RT-PCRprimer 1B 29 cagaacttca gcacacacag gaac 24 30 25 DNA Artificial Sequenceprimer 30 atatcgccgc gctcgtcgtc gacaa 25 31 26 DNA Artificial Sequenceprimer 31 agccacacgc agctcattgt agaagg 26 32 15 PRT Homo sapiens 32 TyrGln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp Ile Glu His 1 5 10 15

1. An isolated STRAP-1 protein having an amino acid sequence shown in FIG. 1A (SEQ ID NO. XX).
 2. An isolated polypeptide of at least 15 contiguous amino acids of the protein of claim
 1. 3. An isolated polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence shown in FIG. 1A (SEQ ID NO. XX) over its entire length.
 4. An isolated polynucleotide selected from the group consisting of (a) a polynucleotide having the sequence as shown in FIG. 1A (SEQ ID NO. XX), wherein T can also be U; (b) a polynucleotide encoding a STRAP-1 polypeptide whose sequence is encoded by the cDNA contained in plasmid 8P1D4 clone 10.1 as deposited with American Type Culture Collection as Accession No. 98849; and (c) a polynucleotide encoding the STRAP-1 protein of claim
 1. 5. An isolated polynucleotide which is fully complementary to a polynucleotide according to claim
 4. 6. A recombinant expression vector which contains a polynucleotide according to claim
 4. 7. A host cell which contains an expression vector according to claim
 6. 8. A process for producing a STRAP-1 protein comprising culturing a host cell of claim 7 under conditions sufficient for the production of the polypeptide and recovering the STRAP-1 protein from the culture.
 9. A STRAP-1 polypeptide produced by the method of claim
 8. 10. An isolated STRAP-2 protein comprising the amino acid sequence shown in FIG. 9 (SEQ ID NO. XX).
 11. An isolated polypeptide of at least 15 contiguous amino acids of the protein of claim
 10. 12. An isolated polynucleotide selected from the group consisting of (a) a polynucleotide having the sequence as shown in FIG. 9 (SEQ ID NO. XX), wherein T can also be U; and (b) polynucleotide encoding the STRAP-2 protein of claim
 10. 13. An isolated polynucleotide which is fully complementary to a polynucleotide according to claim
 12. 14. An antibody which (a) immunohistochemically stains 293T cells transfected with an expression plasmid encoding STRAP-1 according to claim 1, wherein the transfected 293T cells express STRAP-1 protein; and, (b) does not immunohistochemically stain untransfected 293T cells.
 15. The antibody of claim 14, wherein the 293T cells are transfected with an expression plasmid containing the STRAP-1 coding sequence within plasmid 8P1D4 clone 10.1 as deposited with American Type Culture Collection as Accession No.
 98849. 16. An antibody which immunospecifically binds to the STRAP-1 protein of claim 1 or the polypeptide of claim
 2. 17. A monoclonal antibody according to claim
 16. 18. A fragment of the antibody of claim
 17. 19. A recombinant protein comprising the antigen binding domain of the antibody of claim
 17. 20. The antibody of claim 17 which is labeled with a detectable marker.
 21. The monoclonal antibody of claim 17 which is conjugated to a toxin.
 22. The monoclonal antibody of claim 17 which is conjugated to a therapeutic agent.
 23. The antibody fragment of claim 18 which is labeled with a detectable marker.
 24. The recombinant protein of claim 19 which is labeled with a detectable marker.
 25. An antibody which immunospecifically binds to the STRAP-2 protein of claim 10 or the polypeptide of claim
 11. 26. A monoclonal antibody according to claim
 25. 27. The antibody of claim 26 which is labeled with a detectable marker.
 28. The monoclonal antibody of claim 26 which is conjugated to a toxin.
 29. The monoclonal antibody of claim 26 which is conjugated to a therapeutic agent.
 30. An assay for detecting the presence of a STRAP-1 protein in a biological sample comprising contacting the sample with an antibody of claim 20, an antibody fragment of claim 23, or a recombinant protein of claim 24, and detecting the binding of STRAP-1 protein in the sample thereto.
 31. An assay for detecting the presence of a STRAP-2 protein in a biological sample comprising contacting the sample with an antibody of claim 27, and detecting the binding of STRAP-2 protein in the sample thereto.
 32. An assay for detecting the presence of a STRAP-1 polynucleotide in a biological sample, comprising (a) contacting the sample with a polynucleotide probe which specifically hybridizes to the STRAP-1 cDNA contained within plasmid 8P1D4 clone 10.1 as deposited with American Type Culture Collection as Accession No. 98849, or the polynucleotide as shown in FIG. 1A (SEQ ID NO. XX), or the complements thereof; and (b) detecting the presence of a hybridization complex formed by the hybridization of the probe with STRAP-1 polynucleotide in the sample, wherein the presence of the hybridization complex indicates the presence of STRAP-1 polynucleotide within the sample.
 33. An assay for detecting the presence of a STRAP-2 polynucleotide in a biological sample, comprising (a) contacting the sample with a polynucleotide probe which specifically hybridizes to a polynucleotide of claim 12 or its complement; and (b) detecting the presence of a hybridization complex formed by the hybridization of the probe with STRAP-2 polynucleotide in the sample, wherein the presence of the hybridization complex indicates the presence of STRAP-2 polynucleotide within the sample.
 34. An assay for detecting the presence of STRAP-1 mRNA in a biological sample comprising: (a) producing cDNA from the sample by reverse transcription using at least one primer; (b) amplifying the cDNA so produced using STRAP-1 polynucleotides as sense and antisense primers to amplify STRAP-1 cDNAs therein; (c) detecting the presence of the amplified STRAP-1 cDNA, wherein the STRAP-1 polynucleotides used as the sense and antisense probes are capable of amplifying the polynucleotide shown in FIG. 1A (SEQ ID NO. XX).
 35. An assay for detecting the presence of STRAP-2 mRNA in a biological sample comprising: (a) producing cDNA from the sample by reverse transcription using at least one primer; (b) amplifying the cDNA so produced using STRAP-2 polynucleotides as sense and antisense primers to amplify STRAP-2 cDNAs therein; (c) detecting the presence of the amplified STRAP-2 cDNA, wherein the STRAP-2 polynucleotides used as the sense and antisense probes are capable of amplifying the polynucleotide shown in FIG. 9 (SEQ ID NO. XX).
 36. A composition for the treatment of prostate cancer comprising an antibody according to claim 17, 21 or 22, wherein the antibody binds to an extracellular domain of STRAP-1.
 37. A composition for the treatment of colon cancer comprising an antibody according to claim 17, 21 or 22, wherein the antibody binds to an extracellular domain of STRAP-1.
 38. A composition for the treatment of bladder cancer comprising an antibody according to claim 17, 21 or 22, wherein the antibody binds to an extracellular domain of STRAP-1.
 39. A composition for the treatment of prostate cancer comprising an antibody according to claim 26 or 28, wherein the antibody binds to an extracellular domain of STRAP-2. 